샤페론(열충격단백질) 기능 못하는 단백질 항상성유지 기능.. 2015 nature

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Nat Rev Mol Cell Biol

. Author manuscript; available in PMC: 2015 Feb 25.

Published in final edited form as: Nat Rev Mol Cell Biol. 2013 Sep 12;14(10):630–642. doi: 10.1038/nrm3658

Chaperone machines for protein folding, unfolding and disaggregation

Helen Saibil 1

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PMCID: PMC4340576  EMSID: EMS61973  PMID: 24026055

The publisher's version of this article is available at Nat Rev Mol Cell Biol

Abstract

Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression‎ in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20–30 kDa domains over distances of 20–50 Å and rotations of up to 100°.

분자 샤프론은 

초기 단백질이 고유한 접힘에 도달하도록 돕고, 

복합체 조립 중 열 충격으로부터 하위 유닛을 보호하며, 

단백질 응집을 방지하거나 표적 접힘과 분해를 매개하도록 진화한 

다양한 다중 도메인 단백질 제품군입니다. 

스트레스에 대한 반응으로 발현이 증가하는 것은 

세포의 건강과 유기체의 수명에 중요한 요소입니다. 

활성 부위가 정밀하고 미세하게 조정된 효소와 달리 

샤프론은 다양한 기질에서 작동하는 

강력한 분자 기계입니다. 

샤프론의 작용 메커니즘의 구조적 기반은 밝혀지고 있으며

(특히 열충격 단백질 HSP60, HSP70, HSP90 및 HSP100의 경우), 

일반적으로 20~50Å의 거리와 최대 100°의 회전에 걸쳐

 20~30kDa 도메인의 대규모 변위를 수반하는 것으로 알려져 있습니다.

---

Protein quality control, also known as proteostasis, constitutes the regulation of protein synthesis, folding, unfolding and turnover. It is mediated by chaperone and protease systems, together with cellular clearance mechanisms such as autophagy and lysosomal degradation. These quality control systems have an essential role in the life of cells, ensuring that proteins are correctly folded and functional at the right place and time1,2. They are crucial for mitigating the deleterious effects of protein misfolding and aggregation, which, by unclear mechanisms, can cause cell death in neurodegeneration and other incurable protein misfolding diseases (BOX 1). A set of protein families termed molecular chaperones assists various processes involving folding, unfolding and homeostasis of cellular proteins. After protein denaturation caused by stress (for example, due to heat or toxin exposure) or disease conditions, proteins can be unfolded, disaggregated and then refolded, or they can be targeted for disposal by proteolytic systems. Found in all cellular compartments, chaperones act on a broad range of non-native substrates. The endoplasmic reticulum (ER), in particular, is a major site for protein production and quality control in membrane and secretory systems. If it is overburdened by misfolded proteins, the unfolded protein response (UPR) triggers cell death by apoptosis3.

프로테오스타시스라고도 하는 단백질 품질 관리는

단백질 합성, 접힘, 접힘 해제 및 회전율의 조절을 구성합니다.

이는 자가포식 및 리소좀 분해와 같은

세포 제거 메커니즘과 함께

샤페론 및 프로테아제 시스템에 의해 매개됩니다.

이러한 품질 관리 시스템은

세포의 수명에 필수적인 역할을 하며

단백질이 올바른 위치와 시간에 올바르게 접히고 기능하도록 보장합니다1,2.

이는 불분명한 메커니즘으로 인해

신경 퇴화 및 기타 난치성 단백질 오접힘 질환에서

세포 사멸을 유발할 수 있는 단백질 오접힘 및 응집의

해로운 영향을 완화하는 데 매우 중요합니다(박스 1).

분자 샤프론이라고 불리는 일련의 단백질군은

세포 단백질의 접힘,

펼침 및 항상성 유지와 관련된 다양한 과정을 지원합니다.

스트레스(예: 열 또는 독소 노출) 또는

질병으로 인한 단백질 변성 후,

단백질은 펼쳐지거나 분해되었다가 다시 접히거나

단백질 분해 시스템에 의해 폐기 대상이 될 수 있습니다.

모든 세포 구획에서 발견되는 샤프론은

광범위한 비특이적 기질에 작용합니다.

특히 소포체(ER)는

막 및 분비 시스템에서 단백질 생산과 품질 관리를 위한 주요 부위입니다.

잘못 접힌 단백질로 인해 과부하가 걸리면,

세포 사멸 반응(UPR)이 일어나

세포 사멸을 유발합니다3.

Box 1. Protein misfolding diseases.

Mutations that destabilize a protein can cause the loss of protein function. If the protein is degraded and aggregation is prevented, serious pathological consequences may be avoided. However, the aggregation of misfolded proteins creates toxicity (toxic gain of function). Simple loss-of-function mutations in CFTR (cystic fibrosis transmembrane conductance regulator) destabilize the protein, leading to its misfolding in the endoplasmic reticulum (ER) and subsequent degradation, but they do not cause cell death. Conversely, retinitis pigmentosa mutations in the highly abundant photoreceptor protein rhodopsin affects its folding and transport and eventually result in photoreceptor cell death and blindness111,112.

Serious neurodegenerative conditions, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and prion disease, result from the aggregation of a diverse set of peptides and proteins associated with the conversion to amyloid-like fibrillar assemblies. Although neurodegenerative diseases present an obvious burden in ageing societies, systemic conditions involving amyloids such as type II diabetes are equally serious. The common structural feature of amyloid is its cross β-fold in which the protein, whatever its native structure, is converted into a largely or wholly β-strand form. Short strands stack into ribbons that wind into fibrils with the strands running perpendicular to the fibril axis113–115.

Although the structural and mechanistic basis of cytotoxicity remain obscure, there is evidence for membrane damage by oligomeric intermediates in amyloidogenesis, in addition to overload of protein quality control systems. In healthy individuals, chaperones prevent or rescue cells from pathological consequences by promoting refolding, degradation or sequestration into non-toxic aggregates116–119.

The insulin-like signalling pathways that regulate lifespan provide a link between ageing and loss of proteostasis capacity1,2. The role of chaperones in these processes has prompted efforts to chemically modulate these systems, with the goal of providing global protection against protein misfolding120,121.

박스 1. 단백질 오접힘 질환.

단백질을 불안정하게 만드는 돌연변이는

단백질 기능의 상실을 초래할 수 있습니다.

단백질이 분해되고 응집이 방지되면

심각한 병리학적인 결과를 피할 수 있습니다.

그러나

잘못 접힌 단백질의 응집은

독성(독성 기능 증가)을 유발합니다.

CFTR(낭포성 섬유증 막 통과 전도도 조절인자)의 단순한 기능 상실 돌연변이는 단백질을 불안정하게 만들어 소포체(ER)에서의 잘못된 폴딩과 후속 분해로 이어지지만 세포 사멸은 일으키지 않습니다. 반대로, 고농도 광수용체 단백질 로돕신의 망막색소변성증 돌연변이는 접힘과 수송에 영향을 미치고 결국 광수용체 세포 사멸과 실명을 초래합니다111,112.

알츠하이머병, 파킨슨병, 헌팅턴병, 프리온병 등

심각한 신경 퇴행성 질환은 아밀로이드 유사 섬유소 집합체로의 전환과 관련된

다양한 펩타이드 및 단백질의 응집으로 인해 발생합니다.

신경 퇴행성 질환은 고령화 사회에서 분명한 부담이 되지만,

제2형 당뇨병과 같은 아밀로이드와 관련된 전신 질환도 똑같이 심각한 문제입니다.

아밀로이드의 일반적인 구조적 특징은

단백질이 원래의 구조와 상관없이 대부분 또는 전체적으로 β-가닥 형태로 전환되는 교차 β-배열입니다.

짧은 가닥이 리본으로 쌓여 피브릴로 감기고 가닥이 피브릴 축에 수직을 이루면서113-115.

세포 독성의 구조적 및 기계적인 근거는 아직 불분명하지만,

아밀로이드 생성에서 올리고머 중간체에 의한 막 손상과

단백질 품질 관리 시스템의 과부하에 대한 증거가 있습니다.

건강한 사람의 경우,

샤프론은 재접합, 분해 또는 비독성 응집체로의 격리를 촉진하여

세포를 병리학적인 결과로부터 예방하거나 구출합니다116-119.

수명을 조절하는 인슐린 유사 신호 경로는

노화와 단백질 유지 능력 상실 사이의 연관성을 제공합니다1,2.

이러한 과정에서

샤페론의 역할은 단백질 오폴딩에 대한 전반적인 보호를 제공하는 것을 목표로

이러한 시스템을 화학적으로 조절하려는 노력을

촉발시켰습니다120,121.

Chaperones are not typical macromolecular machines with a well-defined substrate. The major molecular chaperones (TABLE 1) have little specificity but provide essential assistance to a complex and highly specific process, protein folding4,5. How do they assist folding or unfolding of diverse proteins? Most of the main chaperones use cycles of ATP binding and hydrolysis to act on non-native polypeptides, facilitating their folding or unfolding6. Others simply have a ‘handover’ role, protecting nascent subunits during the assembly process. Some ATP-dependent chaperones, also known as protein remodelling factors, mediate targeted disassembly, unfolding or even reversal of aggregation. Because of the disordered nature of unfolded, partially folded or aggregated proteins, structural details are lacking for the interactions between chaperones and their protein substrates.

샤페론은 잘 정의된 기질을 가진 전형적인 거대 분자 기계가 아닙니다.

주요 분자 샤페론(표 1)은

특이성이 거의 없지만

복잡하고 매우 특정한 과정인

단백질 폴딩4,5 에 필수적인 도움을 제공합니다.

이들은 어떻게 다양한 단백질의 접힘 또는 펼침을 보조할까요?

대부분의 주요 샤프론은

ATP 결합 및 가수분해 주기를 사용하여 비네이티브 폴리펩타이드에 작용하여

폴딩 또는 폴딩을 촉진합니다6.

다른 샤프론은

단순히 조립 과정에서 초기 서브유닛을 보호하는

'핸드오버 handover' 역할을 합니다.

단백질 리모델링 인자로도 알려진

일부 ATP 의존성 샤프론은

표적 분해, 전개 또는 응집의 역전까지 매개합니다.

펼쳐지거나 부분적으로 접히거나 응집된 단백질의 무질서한 특성으로 인해

샤프론과 단백질 기질 간의 상호 작용에 대한 구조적 세부 정보가 부족합니다.

표 1. ATP 의존성 샤프론, 보조 인자 및 기능의 예.

Table 1. ATP-dependent chaperones, examples of their cofactors and functions.

Chaperones*CofactorsFunctions

Chaperonins
HSP60 (also known as CPN60), GroEL (Escherichia coli), CCT (mammals), thermosome (archaea)	HSP10 (also known as CPN10), GroES, prefoldin	Protein folding, prevention of aggregation
HSP70 system
DnaK (E. coli), Ssa, Ssb (Saccharomyces cerevisiae), BiP (also known as GRP78) (mammals; ER)	HSP40, DnaJ, Sis1, Hdj1, NEFs, GrpE, HSP110	Unfolding, disaggregation, stabilization of extended chains, translocation across organelle membranes, folding, regulation of the heat-shock response, targeting substrates for degradation
HSP90 system
HptG (E. coli), GRP94 (ER)	HOP, p50, AHA1, p23, FKPB52, UNC45	Binding, stabilization and maturation of steroid receptors and protein kinases, delivery to proteases, buffer for genetic variation, regulation of substrate selection and fate, myosin assembly
HSP100
ClpA, ClpB, ClpX, HslU (bacteria; mitochondria and chloroplasts), p97, RPT1–RPT6 (eukaryotic)	HSP70 system, ClpP, ClpS	Unfolding, proteolysis, thermotolerance, resolubilization of aggregates, remodelling

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AHA1, activator of HSP90 ATPase 1; BiP, binding immunoglobulin protein; CCT, chaperonin-containing TCP1 complex; ER, endoplasmic reticulum; FKBP52, 52 kDa FK506-binding protein; GRP78, 78 kDa glucose-regulated protein; HSP, heat shock protein; HOP, HSC70–HSP90-organizing protein; NEFs, nucleotide exchange factors.

*

The species and/or localization is specified in brackets.

An emerging functional feature of chaperones is their highly dynamic behaviour. Despite the great importance and utility of X-ray crystal structures, the resulting atomic structures can give a misleading impression of static, fixed conformations. It seems that the conformations of these ATPases are only weakly coupled to their nucleotide states (that is, whether they are bound to ATP, ADP or in the unbound state) and that they are in a continual state of rapid fluctuation.

This Review focusses on the roles and mechanisms of representatives of the major families of general, ATP-dependent chaperones, namely the heat shock proteins (HSPs; also known as stress proteins) HSP60, HSP70, HSP90 and HSP100. We summarize our current understanding of these allosteric machines and address the ways in which the energy of ATP binding and hydrolysis are used to unfold misfolded polypeptides for either refolding or disaggregation. These considerations underlie a key unanswered question: which protein conformations have chaperones evolved to prevent (under conditions of stress or in misfolding diseases), that is, what is the nature of the cytotoxic species that result when protein homeostasis fails?

종 및/또는 위치는 괄호 안에 명시되어 있습니다.

샤프론의 새로운 기능적 특징은 매우 역동적인 동작입니다. 

X-선 결정 구조의 중요성과 유용성에도 불구하고, 

결과물인 원자 구조는 정적이고 고정된 형태라는 잘못된 인상을 줄 수 있습니다. 

이러한 ATPase의 형태는 뉴클레오티드 상태

(즉, ATP, ADP에 결합되어 있는지 또는 결합되지 않은 상태인지 여부)에 따라 약하게 결합되어 있으며 

지속적으로 빠르게 변동하는 상태인 것으로 보입니다.

이 리뷰에서는 

일반적인 ATP 의존성 샤프론의 주요 계열, 즉 열충격 단백질(HSP; 스트레스 단백질이라고도 함) HSP60, HSP70, HSP90 및 HSP100의 역할과 메커니즘에 초점을 맞추고 있습니다. 

우리는 

이러한 보조 기계에 대한 현재 이해를 요약하고

 ATP 결합과 가수분해의 에너지가 잘못 접힌 폴리펩타이드를 

재접힘 또는 분해하는 데 사용되는 방식을 다룹니다

. 이러한 고려 사항은 

스트레스 조건 또는 오폴딩 질환에서 

어떤 단백질 형태를 방지하기 위해 샤프론이 진화했는지, 

즉 단백질 항상성이 실패할 때 발생하는 세포 독성 종의 특성은 무엇인지에 대한 

핵심적인 미답 질문의 근간이 됩니다.

Chaperone families

Members of the HSP60 (known as GroEL in Escherichia coli), HSP70 (known as DnaK in E. coli), HSP90 (known as HptG in E. coli) and HSP100 (known as ClpA and ClpB in E. coli) families (the number indicates the molecular mass of each HSP subunit) interact either with aggregation-prone, non-native polypeptides or with proteins tagged for degradation.

HSP70 coordinates cellular functions by directing substrates for unfolding, disaggregation, refolding or degradation. HSP90 integrates signalling functions, acting at a late stage of folding of substrates that are important in cellular signalling and development and targeting substrates for proteolysis. By contrast, HSP60 acts at early stages of folding and provides an outstanding example of a highly coordinated and symmetric allosteric machine for protein folding. HSP100 is a sequential ‘threading’ machine for unfolding that cooperates with either a protease ring for degradation or HSP70 for disaggregation, thus avoiding the toxic effects of aggregation.

The mechanisms of action and allostery of the HSP60 and HSP70 families are understood in some detail. HSP60 forms symmetrical, self-contained complexes in which the substrate- and nucleotide-binding sites are located inside cavities, and they act in a concerted and global way on the substrate. By contrast, HSP70 exposes regulatory surfaces and cooperates with various binding proteins that can redirect its activity. It acts locally on unfolded regions of the substrate polypeptide.

HSP70 and HSP90 are highly interactive, functioning with many partners and cofactors. Conversely, HSP60 and HSP100 are ‘loners’. They have few interacting partners and their active sites are not exposed on the outer surface of the protein complex. Despite their very different modes of action, these general chaperones share the common property of binding various non-native proteins to prevent their aggregation.

샤프론 계열

HSP60(대장균에서는 GroEL로 알려져 있음), 

HSP70(대장균에서는 DnaK로 알려져 있음), HSP90(대장균에서는 HptG로 알려져 있음) 및 

HSP100(대장균에서는 ClpA 및 ClpB로 알려져 있음) 계열의 구성원

(숫자는 각 HSP 하위 단위의 분자량을 나타냄)은 

응집하기 쉬운 비네이티브 폴리펩티드 또는 

분해하도록 태그된 단백질과 상호작용을 합니다.

HSP70은 

전개, 분해, 재접합 또는 분해를 위해 기질을 지시하여 

세포 기능을 조정합니다. 

HSP90은 

신호 기능을 통합하여 세포 신호 및 발달에 중요한 기질의 접힘 후기 단계에서 작용하고 

단백질 분해를 위해 기질을 표적으로 삼습니다. 

이와 대조적으로 HSP60은 폴딩의 초기 단계에서 작용하며 단백질 폴딩을 위한 고도로 조정되고 대칭적인 알로스테릭 기계의 뛰어난 예를 제공합니다. HSP100은 단백질 분해를 위해 프로테아제 링과 협력하거나 분해를 위해 HSP70과 협력하여 응집의 독성 영향을 피하는 순차적 '나사산' 접힘 기계입니다.

HSP60과 HSP70 계열의 작용 메커니즘과 알로스테리는 어느 정도 이해되고 있습니다. HSP60은 기질과 뉴클레오타이드 결합 부위가 공동 내부에 위치한 대칭적이고 독립적인 복합체를 형성하며, 기질에 대해 전체적으로 조화롭게 작용합니다. 이와 대조적으로 HSP70은 조절 표면을 노출하고 다양한 결합 단백질과 협력하여 활성을 전환할 수 있습니다. 이는 기질 폴리펩타이드의 펼쳐진 영역에서 국소적으로 작용합니다.

HSP70과 HSP90은 상호 작용이 매우 활발하여 많은 파트너 및 보조 인자와 함께 작용합니다. 반대로 HSP60과 HSP100은 '외톨이'입니다. 이들은 상호 작용하는 파트너가 거의 없고 활성 부위가 단백질 복합체의 외부 표면에 노출되어 있지 않습니다. 작용 방식은 매우 다르지만, 이러한 일반적인 샤프론은 다양한 비 고유 단백질과 결합하여 응집을 방지한다는 공통점을 공유합니다.

HSP70 — a tuneable chaperone system

HSP70 is the most abundant chaperone and exists as many orthologues in different cellular compartments. In association with various cofactors it carries out diverse functions, including protein folding, trans location across organelle membranes and disaggregation of aggregates. HSP70 has two domains: an ATPase domain and a substrate-binding domain. Its activity depends on dynamic interactions between these two domains and also on interactions between these domains and co-chaperones such as the HSP40 proteins (also known as J proteins, named after E. coli DnaJ) and nucleotide exchange factors (NEFs, which stimulate ADP release and nucleotide exchange after ATP hydrolysis)6–8.

Cellular functions of HSP70

Even transient binding of an extended segment of a polypeptide chain to HSP70 could prevent misfolding and aggregation and maintain the substrate in an unfolded state for translocation to another cellular compartment. Indeed, the HSP70 system is an important component of the organelle translocation system on both sides of the membrane. The conformational cycle of HSP70 is used both for delivery of the substrate protein to the translocase that transports it across the organelle membrane and to capture or pull on the translocated polypeptide (reviewed in REF. 9). Regarding folding from the unfolded state, it seems likely that polypeptides can collapse into their native fold in free solution upon release from HSP70. Failure to reach the correctly folded state would lead to re-binding. Thus, the role of HSP70 in folding seems to be stabilizing the unfolded state or unfolding proteins until they can spontaneously fold upon reaching their correct cellular destination10.

In addition to its role in folding, HSP70 has other specific cellular functions. For example, together with auxilin (which is also a J protein co-chaperone), it disassembles the clathrin coat on membrane vesicles after completion of clathrin-mediated endocytosis11. It also cooperates with HSP100 ATPases in disaggregating large aggregates (see below). The corresponding partner of HSP70 for disaggregation and/or detoxification of aggregates in the cytosol of higher eukaryotes has recently been identified as the NEF HSP110, which also has chaperone activity12–15.

HSP70 - 조정 가능한 샤프론 시스템

HSP70은

가장 풍부한 샤프론이며

여러 세포 구획에 많은 유사체가 존재합니다.

다양한 보조 인자와 연계하여 단백질 폴딩, 세포막을 가로지르는 트랜스 위치, 응집체 분해 등 다양한 기능을 수행합니다. HSP70은 ATPase 도메인과 기질 결합 도메인의 두 가지 도메인을 가지고 있습니다. 이 두 도메인 간의 역동적인 상호작용과 이 두 도메인과 HSP40 단백질(대장균 DnaJ의 이름을 딴 J 단백질이라고도 함) 및 뉴클레오티드 교환 인자(NEF, ATP 가수분해 후 ADP 방출 및 뉴클레오티드 교환을 자극하는)6-8 와 같은 공동 샤페론 간의 상호작용에 따라 활성이 달라집니다.

HSP70의 세포 기능

폴리펩타이드 사슬의 확장된 세그먼트가 HSP70에 일시적으로 결합하더라도 잘못된 접힘과 응집을 방지하고 기질을 펼쳐진 상태로 유지하여 다른 세포 구획으로 전위를 유도할 수 있습니다. 실제로 HSP70 시스템은 세포막 양쪽의 세포소기관 전위 시스템의 중요한 구성 요소입니다. HSP70의 형태 주기는 기질 단백질을 세포막을 가로질러 운반하는 트랜스로케이스로 전달하고 전위된 폴리펩타이드를 포획하거나 잡아당기는 데 모두 사용됩니다( 참고 9 참조). 펼쳐진 상태에서 접는 것과 관련하여, 폴리펩타이드는 HSP70에서 방출되면 자유 용액에서 원래의 접힌 상태로 붕괴될 수 있는 것으로 보입니다. 올바르게 접힌 상태에 도달하지 못하면 재결합으로 이어질 수 있습니다. 따라서 폴딩에서 HSP70의 역할은 단백질이 올바른 세포 목적지에 도달하면 자발적으로 접힐 수 있을 때까지 펼쳐진 상태 또는 펼쳐진 단백질을 안정화시키는 것으로 보입니다10.

HSP70은 접는 역할 외에도 다른 특정 세포 기능을 수행합니다. 예를 들어, J 단백질 공동 샤페론이기도 한 옥실린과 함께 클라트린 매개 세포 내분비가 완료된 후 막 소포의 클라트린 외피를 분해합니다11. 또한 큰 응집체를 분해할 때 HSP100 ATPase와 협력합니다(아래 참조). 고등 진핵생물의 세포질에서 응집체의 분해 및/또는 해독을 위한 HSP70의 상응하는 파트너는 최근 샤프론 활성을갖는 NEF HSP110으로 확인되었습니다12 -15.

Structural basis of HSP70 function

The atomic structures of the ATPase domain and the substrate-binding domain of HSP70 were determined separately in the 1990s. Unexpectedly, the ATPase domain was found to have the same fold as actin and hexokinase, with two flexible domains surrounding a deep, nucleotide-binding cleft that closes around ATP16,17 (FIG. 1a). The substrate-binding domain is thin and brick-shaped with a cleft capped by a mobile α-helical lid. Both the lid and the cleft open to allow substrate binding, which can then be trapped by the closing lid18 (FIG. 1a). The nucleotide state of the ATPase domain affects the opening (stimulated by ATP binding) and shutting (after ATP hydrolysis) of the substrate-binding site. However, the two domains, which are connected by a flexible linker, are not seen together in most crystal structures. The flexible linker, located at the base of the two domains remote from the cleft opening, is a key site in allosteric regulation.

HSP70 기능의 구조적 기초

ATPase 도메인과 HSP70의 기질 결합 도메인의 원자 구조는

1990년대에 개별적으로 결정되었습니다.

예상외로

ATPase 도메인은 액틴 및 헥소키나아제와 동일한 접힘을 가지고 있으며,

두 개의 유연한 도메인이 ATP16,17 주변에서 닫히는 깊은 뉴클레오티드 결합 틈새를 둘러싸는 것으로 밝혀졌습니다(그림 1a).

기질 결합 도메인은 얇고 벽돌 모양이며 이동식 α 나선형 뚜껑으로 덮인 틈새가 있습니다.

뚜껑과 틈새는 모두 열려 기질 결합을 허용하며, 닫힌 뚜껑18에 의해 갇힐 수 있습니다(그림 1a).

ATPase 도메인의 뉴클레오티드 상태는

기질 결합 부위의 개방(ATP 결합에 의해 자극됨) 및 폐쇄(ATP 가수분해 후)에 영향을 미칩니다.

그러나 유연한 링커로 연결된 두 도메인은 대부분의 결정 구조에서 함께 보이지 않습니다.

갈라진 구멍에서 멀리 떨어진 두 도메인의 기저부에 위치한 플렉시블 링커는 알로스테릭 조절의 핵심 부위입니다.

Figure 1. HSP70 assemblies.

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a ∣ In the ADP-bound or nucleotide-free state, the nucleotide-binding domain (green; Protein Data Bank (PDB) code: 3HSC)16 of heat shock protein 70 (HSP70) is connected by a flexible linker to the substrate-binding domain (blue; PDB code: 1DKZ), with the lid domain (red) locking a peptide substrate (yellow) into the binding pocket18. A side view of the substrate domain is shown on the right. A cartoon depicting the two-domain complex is shown below. The bound nucleotide is shown in space filling format. b ∣ In the ATP-bound state, the lid opens, and both the lid and the substrate-binding domain dock to the nucleotide-binding domain (PDB code: 4B9Q)20. The corresponding cartoon of this conformation is shown below. When ATP binds, the cleft closes, triggering a change on the outside of the nucleotide-binding domain that creates a binding site for the linker region. Linker binding causes the substrate-binding domain and the lid domain to bind different sites on the nucleotide-binding domain, resulting in a widely opened substrate-binding site that enables rapid exchange of polypeptide substrates. After hydrolysis, the domains separate and the lid closes over the bound substrate. Such binding and release of extended regions of polypeptide chain are thought to unfold and stabilize non-native proteins either for correct folding or degradation.

a ∣ ADP 결합 또는 뉴클레오타이드가 없는 상태에서 뉴클레오티드 결합 도메인(녹색, 단백질 데이터 뱅크(PDB) 코드): 3HSC)16은 열충격 단백질 70(HSP70)의 기질 결합 도메인(파란색; PDB 코드: 1DKZ)에 유연한 링커로 연결되어 있으며, 뚜껑 도메인(빨간색)은 펩타이드 기질(노란색)을 결합 포켓18에 고정합니다. 기질 도메인의 측면도는 오른쪽에 나와 있습니다. 두 도메인 복합체를 묘사한 만화는 아래에 나와 있습니다. 결합된 뉴클레오티드는 공간 채우기 형식으로 표시되어 있습니다.

b ∣ ATP 결합 상태에서는 뚜껑이 열리고 뚜껑과 기질 결합 도메인이 모두 뉴클레오티드 결합 도메인(PDB 코드: 4B9Q)20 에 도킹됩니다. 이 구조의 해당 만화는 아래와 같습니다. ATP가 결합하면 틈새가 닫히면서 뉴클레오티드 결합 도메인의 외부에 변화를 일으켜 링커 영역의 결합 부위가 만들어집니다. 링커 결합은 기질 결합 도메인과 뚜껑 도메인이 뉴클레오티드 결합 도메인의 다른 부위에 결합하게 하여 넓게 열린 기질 결합 부위를 만들어 폴리펩티드 기질을 빠르게 교환할 수 있게 합니다. 가수분해 후에는 도메인이 분리되고 뚜껑이 결합된 기질 위로 닫힙니다. 이러한 폴리펩타이드 사슬의 확장된 영역의 결합과 방출은 올바른 접힘 또는 분해를 위해 비네이티브 단백질을 펼치고 안정화시키는 것으로 생각됩니다.

A first view of the domain interaction came from the structure of yeast Sse1 (a homologue of mammalian HSP110)19. Although Sse1 and HSP110 are structural homologues of HSP70, they act as HSP70 NEFs. More recently, the crystal structure of a disulphide-trapped form of ATP-bound DnaK (which is the E. coli homologue of mammalian HSP70), together with methyl transverse relaxation optimized spectroscopy (methyl TROSY) nuclear magnetic resonance (NMR) and mutational probing of the domain association in a mutant deficient in ATP hydrolysis revealed how the HSP70 domains interact20–22. Unlike Sse1 or HSP110, DnaK is highly dynamic, with rapid fluctuations between the docked and free conformations in all nucleotide-bound states.

A remarkable feature of the domain-docked complex is the intimate association of the substrate-binding domain with the ATPase domain. The substrate-binding domain is almost turned inside-out to wrap around the ATPase domain, with an extremely open orientation of its helical lid and a scissor-like motion of its β-subdomain that opens up the peptide-binding cleft (FIG. 1b). It has been proposed that the HSP70 mechanism of action involves several key steps. First, allosteric signalling from ATP binding and closure of the nucleotide-binding cleft creates a binding site on the ATPase domain for the interdomain linker, which then recruits the substrate-binding domain. This domain docking distorts the substrate-binding cleft and opens the lid, which then binds to a different part of the ATPase domain20–22.

도메인 상호 작용에 대한 첫 번째 견해는 효모 Sse1(포유류 HSP110의 상동체)19 의 구조에서 나왔습니다. Sse1과 HSP110은 HSP70의 구조적 상동체이지만, HSP70 NEF로 작용합니다. 최근에는 이황화 이완 최적화 분광법(메틸 TROSY) 핵자기공명(NMR) 및 ATP 가수분해가 결핍된 돌연변이체에서 도메인 연관성에 대한 돌연변이 프로빙과 함께 이황화물 포획 형태의 ATP 결합 DnaK 결정 구조를 통해 HSP70 도메인이 어떻게 상호작용하는지밝혀졌습니다20 -22. Sse1이나 HSP110과 달리 DnaK는 매우 역동적이며 모든 뉴클레오티드 결합 상태에서 도킹된 형태와 자유 형태 간에 급격한 변동이 있습니다.

도메인 도킹 복합체의 주목할 만한 특징은 기질 결합 도메인과 ATPase 도메인의 밀접한 연관성입니다. 기질 결합 도메인은 나선형 뚜껑의 극도로 열린 방향과 펩타이드 결합 틈새를 여는 β-서브도메인의 가위 같은 움직임으로 ATPase 도메인을 거의 뒤집어 감싸고 있습니다(그림 1b). HSP70의 작용 메커니즘에는 몇 가지 주요 단계가 포함되어 있다고 제안되었습니다. 첫째, ATP 결합과 뉴클레오티드 결합 틈새의 폐쇄로 인한 알로스테릭 신호가 도메인 간 링커를 위한 ATPase 도메인에 결합 부위를 생성하고, 이 부위가 기질 결합 도메인을 모집합니다. 이 도메인 도킹은 기질 결합 틈새를 왜곡하고 뚜껑을 열어 ATPase 도메인의 다른 부분에 결합합니다20-22.

Regulation by co-chaperones

HSP70 acts together with two co-chaperones in protein folding, namely an HSP40 and a NEF. The HSP40 family is very diverse, with many specialized members targeting HSP70 to specific sites or functions7. HSP40 is thought to act as the primary substrate recruiter forHSP70 and stimulates the HSP70 ATPase. For pathways involving nonspecific protein folding and refolding, the general HSP40 is an elongated, V-shaped dimer containing the characteristic, helical J domain that activates the HSP70 ATPase by binding at or near the interdomain linker23,24. The J domain is followed by a disordered, Gly-Phe-rich region, two tandem β-subdomains and a dimerization domain25. One of the β-subdomains contains a surface-exposed substrate-binding site. It seems likely that hydrophobic segments of a substrate polypeptide, following their initial recruitment to the shallow, accessible binding sites of HSP40, are delivered to the deeper channel of HSP70 for binding via the polypeptide backbone, with the J domain stimulating the ATPase26,27,8. Thus, J proteins interact with both the nucleotide- and substrate-binding domains of HSP70, with flexibly linked sites stimulating the ATPase and delivering the bound polypeptide. NEFs such as E. coli GrpE or eukaryotic HSP110 interact near the entrance to the nucleotide cleft, moving the HSP70 subdomain IIb (FIG. 1a) and opening the cleft for nucleotide exchange28–30. Although these interactions have been observed separately, how the dynamic complex functions as a whole has not yet been shown.

공동 샤페론에 의한 조절

HSP70은 단백질 폴딩에서 두 개의 공동 샤프론, 즉 HSP40과 NEF와 함께 작용합니다. HSP40 계열은 매우 다양하며, 특정 부위 또는 기능에 HSP70을 표적으로 하는 특수한 멤버가 많습니다7 . HSP40은 HSP70의 주요 기질 모집자 역할을 하며 HSP70 ATPase를 자극하는 것으로 알려져 있습니다. 비특이적 단백질 폴딩 및 재폴딩과 관련된 경로의 경우, 일반적인 HSP40은 도메인 간 링커23,24 에 결합하여 HSP70 ATPase를 활성화하는 특징적인 나선형 J 도메인을 포함하는 길쭉한 V형 이합체입니다. J 도메인 뒤에는 무질서한 글리-페가 풍부한 영역, 2개의 탠덤 β-서브도메인 및 이량체화 도메인이 있습니다25. β-서브 도메인 중 하나는 표면에 노출된 기질 결합 부위를 포함합니다. 기질 폴리펩타이드의 소수성 세그먼트는 HSP40의 얕고 접근 가능한 결합 부위에 처음 결합한 후 폴리펩타이드 백본을 통해 결합을 위해 HSP70의 더 깊은 채널로 전달되고, J 도메인은 ATPase26,27,8 를 자극하는 것으로 보입니다. 따라서 J 단백질은 HSP70의 뉴클레오티드 및 기질 결합 도메인 모두와 상호 작용하며, 유연하게 연결된 부위는 ATPase를 자극하고 결합된 폴리펩타이드를 전달합니다. 대장균 GrpE 또는 진핵생물 HSP110과 같은 NEF는 뉴클레오티드 틈새 입구 근처에서 상호작용하여 HSP70 하위 도메인 IIb를 이동시키고(그림 1a) 뉴클레오티드 교환을 위해 틈새를 열어줍니다28-30. 이러한 상호 작용은 개별적으로 관찰되었지만 동적 복합체가 전체적으로 어떻게 기능하는지는 아직 밝혀지지 않았습니다.

HSP90 — a cellular signalling hub

HSP90, another highly abundant and ubiquitous chaperone, has diverse biological roles, but its mechanism of action is less well-understood than that of the major chaperones. It is a highly flexible, dynamic protein and, in eukaryotes, has a multitude of interactors that regulate its activities, making it a hub for many pathways31–34. Like other stress proteins, HSP90 is capable of binding non-native polypeptides and preventing their aggregation. It seems to act mainly at the late stages of substrate folding. For example, steroid hormone receptors must bind HSP90 for efficient loading of their steroid ligand. The bacterial form seems to act alone and is not crucial for viability, but the eukaryotic forms and their many co-chaperones are essential. HSP90 is functionally more specialized than the other general chaperones. It is important for maturation of signalling proteins in development and cell division, and its substrates include steroid hormone receptors, kinases and key oncogenic proteins such as the tumour suppressor p53.

An intriguing evolutionary hypothesis proposes that HSP90 acts as a buffer for genetic variation by rescuing mutated proteins with altered properties35. A reservoir of such proteins could serve to improve fitness during evolutionary change. Some experimental support for this idea comes from studies investigating the developmental effects of HSP90 inhibitors on Drosophila melanogaster and Arabidopsis thaliana, and from studies examining the effects of environmental stress in yeast36.

HSP90 - 세포 신호 전달 허브

매우 풍부하고 어디에나 존재하는 또 다른 샤프론인 HSP90은 다양한 생물학적 역할을 하지만 주요 샤프론에 비해 그 작용 메커니즘은 잘 알려지지 않았습니다. 매우 유연하고 역동적인 단백질이며, 진핵생물에서는 활동을 조절하는 다양한 상호 작용자를 가지고 있어 여러 경로의 허브 역할을 합니다31-34. 다른 스트레스 단백질과 마찬가지로 HSP90은 비네이티브 폴리펩티드와 결합하여 이들의 응집을 방지할 수 있습니다. 주로 기질 접힘의 후기 단계에서 작용하는 것으로 보입니다. 예를 들어, 스테로이드 호르몬 수용체는 스테로이드 리간드를 효율적으로 로딩하기 위해 HSP90과 결합해야 합니다. 박테리아 형태는 단독으로 작용하는 것처럼 보이며 생존에 중요하지 않지만, 진핵생물 형태와 그 많은 공동 샤프론은 필수적입니다. HSP90은 다른 일반적인 샤프론보다 기능적으로 더 특화되어 있습니다. 발달과 세포 분열에서 신호 단백질의 성숙에 중요하며 스테로이드 호르몬 수용체, 키나아제, 종양 억제제 p53과 같은 주요 발암성 단백질이 그 기질입니다.

흥미로운 진화 가설에 따르면 HSP90은 특성이 변경된 돌연변이 단백질을 구출하여 유전적 변이에 대한 완충제 역할을 한다고 합니다35. 이러한 단백질의 저장소는 진화적 변화 중에 적합성을 향상시키는 역할을 할 수 있습니다. 이 아이디어에 대한 일부 실험적 뒷받침은 초파리(Drosophila melanogaster)와 애기장대(Arabidopsis thaliana)에 대한 HSP90 억제제의 발달 효과를 조사한 연구와 효모에서 환경 스트레스의 영향을 조사한 연구36에서 나옵니다.

HSP90 in complex with nucleotides and substrates

HSP90 forms a dimer of elongated subunits, with each subunit comprising three domains that are linked by flexible regions. It stably dimerizes through its carboxy-terminal domains and also transiently through its amino-terminal ATPase domain when ATP is bound37 (FIG. 2). HSP90 is extremely dynamic, as it fluctuates rapidly between conformations ranging from an open V-shape to a closed form resembling a pair of cupped hands38. The nucleotide-binding site accommodates a bent conformation of ATP, the binding of which causes transient dimerization of the N-terminal domains, characteristics of the GHKL (gyrase, HSP90, His kinase and MutL) ATPase fold shared with the DNA-unwinding enzyme DNA-gyrase39,40. Specific inhibitors of the HSP90 ATPase have marked effects in development and cancer. Although the HSP90 nucleotide state is only weakly coupled to its conformational change41, the many binding partners of HSP90 influence different steps in the functional cycle. HSP90 action is modulated by co-chaperones and client proteins (the term used for ‘substrates’ in the HSP90 system). In addition, phosphorylation, acetylation and other post-translational modifications affect its functional state32.

뉴클레오타이드 및 기질과 복합체를 이루는 HSP90

HSP90은 길쭉한 서브유닛의 이량체를 형성하며, 각 서브유닛은 유연한 영역으로 연결된 세 개의 도메인으로 구성됩니다. 카르복시 말단 도메인을 통해 안정적으로 이량체화되고 ATP가 결합하면 아미노 말단 ATPase 도메인을 통해 일시적으로 이량체화됩니다37 (그림 2). HSP90은 개방형 V자 형태에서 한 쌍의 컵 모양을 닮은 닫힌 형태에 이르기까지 빠르게 변화하기 때문에 매우 역동적입니다38. 뉴클레오티드 결합 부위는 ATP의 구부러진 형태를 수용하며, 이러한 결합은 DNA 풀림 효소인 DNA-gyrase와 공유하는 GHKL(gyrase, HSP90, His kinase 및 MutL) ATPase 폴드의 특성인 N-말단 도메인의 일시적인 이량체화를 유발합니다39,40. HSP90 ATPase의 특정 억제제는 발달과 암에 뚜렷한 영향을 미칩니다. HSP90 뉴클레오타이드 상태는 형태 변화와 약하게 결합되어 있지만41, HSP90의 많은 결합 파트너는 기능 주기의 여러 단계에 영향을 미칩니다. HSP90의 작용은 공동 샤프론과 클라이언트 단백질(HSP90 시스템에서 '기질'이라는 용어로 사용됨)에 의해 조절됩니다. 또한 인산화, 아세틸화 및 기타 번역 후 변형이 기능 상태에 영향을 미칩니다32.

Figure 2. HSP90 conformations and substrate binding.

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Crystal structures of heat shock protein 90 (HSP90) dimers in an open, unliganded state (Protein Data Bank (PDB) code: 2IOQ)122 (part a), a partly closed, ADP-bound state (PDB code: 2O1V)123 (part b) and in a closed, ATP-bound state (PDB code: 2CG9)37 (part c), are shown, and the amino-terminal domain (green), the middle domain (yellow) and the carboxy-terminal domain (blue) are indcated. The open form shown is Escherichia coli HptG, the partly closed ADP-bound form is the canine endoplasmic reticulum-associated HSP90 homologue GRP94 and the ATP-bound form (shown is the ATP analogue AMP-PNP) is yeast Hsc82 (heat shock cognate 82). Nucleotides are shown in space filling format. ATP favours binding to the closed form (part c), whereas hydrolysis or nucleotide release is favoured by a range of more open states (parts a,b). Opening and closing of the cleft are thought to mediate the action of HSP90 on its substrates, although the mechanisms underlying HSP90 action remain largely unclear. The electron microscopy map of HSP90 in complex with the cofactor p50 and its substrate cyclin-dependent kinase 4 (CDK4) is shown48 (part d). Extra density of the side of this asymmetric complex is attributed to the cofactor and substrate.

열충격 단백질 90(HSP90) 이합체의 개방형 비리간드 상태의 결정 구조(단백질 데이터 뱅크(PDB) 코드: 2IOQ)122 (부분 a), 부분적으로 닫힌 ADP 결합 상태(PDB 코드: 2O1V)123 (부분 b), 닫힌 ATP 결합 상태(PDB 코드: 2CG9)37 (부분 c)가 표시되어 있으며 아미노 말단 도메인(녹색), 중간 도메인(노란색) 및 카르복시 말단 도메인(파란색)이 표시되어 있습니다. 표시된 열린 형태는 대장균 HptG, 부분적으로 닫힌 ADP 결합 형태는 개 소포체 관련 HSP90 동족체 GRP94, ATP 결합 형태(ATP 유사체 AMP-PNP로 표시)는 효모 Hsc82(열충격 동족체 82)입니다. 뉴클레오타이드는 공간 채우기 형식으로 표시되어 있습니다. ATP는 닫힌 형태(부분 c)에 결합하는 것을 선호하는 반면, 가수분해 또는 뉴클레오타이드 방출은 보다 개방된 상태(부분 a,b)에 결합하는 것을 선호합니다. 틈새의 개폐는 HSP90의 기질에 대한 작용을 매개하는 것으로 생각되지만, HSP90 작용의 기본 메커니즘은 아직 명확하지 않습니다. 보조 인자 p50 및 그 기질 사이클린 의존성 키나아제 4(CDK4)와 복합체를 이루는 HSP90의 전자 현미경 지도48 (파트 d)가 나와 있습니다. 이 비대칭 복합체의 측면의 추가 밀도는 보조 인자와 기질에 기인합니다.

Co-chaperones that target HSP90 to specific types of client protein include p50 (also known as CDC37), which recruits kinases and inhibits the ATPase activity of HSP90 (REF. 42). A set of co-chaperones with prolyl isomerase activity, such as the immunophilin 52 kDa FK506-binding protein (FKBP52), are involved in complexes with steroid receptors. These co-chaperones interact through their tetratricopeptide repeat (TPR) domains with a conserved C-terminal motif found on HSP90 and also on HSP70 (REF. 43). Numerous other co-chaperone complexes assemble on HSP90 via TPR domains. For example, HSC70–HSP90-organizing protein (HOP; also known as STI1) recruits HSP70 to HSP90, creating a complex for substrate handover44. Important non-TPR containing co-chaperones include activator of HSP90 ATPase 1 (AHA1) and p23, which is involved in client protein maturation45,37. Together with HSP70, HSP90 also has an important role in targeting substrates for degradation46.

Details of substrate binding to HSP90 are poorly understood. Evidence for substrate-binding sites on all three domains of HSP90 came from low-resolution electron microscopy and mutational studies, which led to a model of hydrophobic surfaces lining the cavity of an open dimer47–50. Despite the lack of a mechanistic understanding of the action of HSP90, specific inhibitors of its ATPase activity, such as geldanamycin, were shown to have important biological effects and form the basis for successful anticancer drugs51.

HSP60 — a protein folding container

Chaperonins (a term specific to this chaperone family) can be divided into two subfamilies: group I is composed of the bacterial chaperonin GroEL and its co-chaperonin GroES, as well as the mitochondrion- and chloroplast-specific HSP60 proteins together with their HSP10 co-chaperonins; and group II chaperonins, which are found in archaea and the eukaryotic cytosol and comprise the archaeal thermosome and eukaryotic CCT (chaperonin-containing TCP1; also known as TriC). In group II, an extra protein domain replaces the group I co-chaperoni n. The bacterial GroEL–GroES chaperonin system is by far the best understood general chaperone.

Chaperonins are self-contained machines that leave little to chance; they provide a complete isolation chamber for protein folding. Early work on bacteriophage assembly, mitochondrial and chloroplast biogenesis led to the realization that related proteins in bacteria, chloroplasts and mitochondria have an essential role in de novo protein folding and assembly as well as refolding stress-denatured proteins52–54.

Chaperonin structures and action

Biochemical, biophysical and structural analyses, particularly of E. coli GroEL–GroES, have revealed many important parts of the mechanism of action55,56. GroEL crystal structures reveal details of the start and end states of extensive movements of this chaperonin through concerted rigidbody rotations of the subunit domains. Unliganded (apo) GroEL forms a 15 nm long cylindrical structure composed of back-to-back rings of seven 60 kDa subunits57 (FIG. 3a). These rings surround open cavities of ~5 nm diameter, the walls of which are lined by a band of continuous hydrophobic surfaces. The two rings alternately go through cycles of ATP binding and hydrolysis. Upon ATP binding, a GroEL ring rapidly recruits the co-chaperonin GroES, a ring of seven 10 kDa subunits, which caps the cavity, entailing a dramatic structural reorganization to convert the open ring into an enclosed chamber with a hydrophilic lining58. In the GroES-bound ring, the substrate-binding apical domains are elevated by 60° and twisted by 90° relative to the unliganded ring.

Figure 3. GroEL conformations and substrate complexes.

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a ∣ Overview of unliganded (apo) GroEL (Protein Data Bank (PDB) code:1OEL)57 (left) and the GroEL–GroES complex (PDB code: 1SVT)58 (right). The overall shapes are shown as blue surfaces, with three subunits coloured by domain in red, green and yellow in apo GroEL. One subunit of GroEL and one of GroES (cyan) are highlighted in the GroEL–GroES complex. b ∣ Conformation of a GroEL subunit in the apo form (left) and the GroES-bound form (right), with GroEL key sites indicated (GroES is not shown). c ∣ Cartoons of complexes with folding proteins. Hydrophobic surfaces and residues are shown in yellow and polar residues in green. d ∣ Cut open view of the cryo-electron microscopy structure (Electron Microscopy Data Bank code: EMD-1548) of GroEL (PDB code: 1AON) in complex with bacteriophage 56 kDa capsid protein (gp31) (PDB code: 1G31), with a non-native gp23 (PDB code: 1YUE) bound to both rings64. The pink density in the folding chamber corresponds to newly folded gp23, and the yellow density in the open ring is part of a non-native gp23 subunit. The corresponding atomic structures are shown embedded in the electron microscopy density map, except for the non-native substrate, which is unknown and only partially visualized owing to disorder. The open ring with its hydrophobic lining is the acceptor state for non-native polypeptides, and binding to multiple sites may facilitate unfolding. ATP and GroES binding to the chaperonin create a protected chamber with a hydrophilic lining that allows the encapsulated protein to fold.

The open, hydrophobic lined ring is the acceptor state that captures non-native polypeptides with exposed hydrophobic surfaces, accounting for the lack of binding specificity of group I chaperonins. The interaction with the substrate can extend over 3–4 adjacent GroEL subunits59,60. The actions of GroEL, ATP and GroES exert mechanical forces on the substrate that potentially result in unfolding of trapped, misfolded proteins61. This culminates in a power stroke that ejects the substrate from the hydrophobic sites and simultaneously traps it inside the GroES-capped hydrophilic chamber for folding62. Once encapsulated, the lack of exposed hydrophobic sites or other partners for aggregation, together with the limited enclosure (~7 nm maximum dimension), blocks further misfolding or aggregation pathways, so that the substrate can either follow a folding pathway determined by its amino acid sequence or remain unfolded. After a slow ATP hydrolysis step, the chamber is re-opened, releasing the protein either committed to final folding and assembly or releasing it in a non-native state that will be recaptured by a chaperonin ring. For substrates that are too large to be encapsulated, GroES may still act allosterically to effect productive release of the substrate from the remote open ring63.

Key to understanding this action is to determine the structures of the intermediate complexes when substrate and ATP have bound and GroES is being recruited. At low to intermediate resolution, substrate binding and GroEL domain movements have been characterized by single particle cryo-electron microscopy (cryo-EM) of various intermediate complexes, using statistical analysis to discriminate multiple three-dimensional structures from images of heterogeneous and dynamic complexes. This approach has yielded structural descriptions of chaperonin complexes at different stages of substrate binding and folding and has enabled analysis of the allosteric machinery60,64,65.

Crystal structures as well as kinetic and mutational studies have revealed key allosteric sites in chaperonins. Each subunit contains three domains connected by flexible hinge points (FIG. 3b). The nucleotide-binding pocket is in the equatorial domain, and helix D runs from an Asp residue coordinating the γ-phosphate site to one of the two inter-ring contacts. In the GroES-bound state, the intermediate domain closes over the ATP pocket, bringing a catalytic Asp residue close to the nucleotide. Within each ring, the subunits are interlinked by salt bridges and act in concert, exhibiting positive cooperativity for ATP binding66. Conversely, the two rings act sequentially, exhibiting negative cooperativity, which is transmitted through the two inter-ring contacts. Hydrophobic sites on the apical domain form the GroES- and substrate-binding sites (FIG. 3c). A mobile loop of GroES binds to the distal part of this site, a region also implicated in substrate binding, leading to the notion that GroES and substrate binding are mutually exclusive. However, there is biochemical evidence, although no direct structural information, for an intermediate state in which GroES and substrate are simultaneously bound to GroEL67,68.

Substrate complexes

Crystal structures of extended peptides bound to the GroEL apical domain occupy the same site as the GroES mobile loop58,69,70. This provides a partial view of how substrates might bind, but electron microscopy studies of GroEL with captured non-native proteins show a preference for binding deeper inside the cavity in the more proximal part of the hydrophobic site60,64. Moreover, electron microscopy structures show how substrates bind to the open ring and how they appear in the folding chamber (FIG. 3d). This enclosure imposes an upper limit of under 60 kDa for protein subunits that can be encapsulated. To accommodate its 56 kDa capsid protein, gp23, bacteriophage T4 encodes its own GroES homologue, gp31, to make the cage slightly taller71. The newly folded large domain of gp23, encapsulated and trapped by using a non-hydrolysable ATP analogue, fills the chamber and distorts it64. A trapped, non-native state of another large substrate protein, RuBisCo (ribulose-1,5-bisphosphat e carboxylase oxygenase), has been visualized by cryo-EM, revealing contacts to apical and equatorial domains72.

ATP complexes and domain movements

How does the binding of ATP detach a non-native protein multivalently bound on the hydrophobic surface, resulting in a free subunit isolated in the folding chamber? The conformation of open GroEL rings in the presence of ATP is extremely dynamic. Sorting of heterogeneous complexes by single particle electron microscopy has resolved a set of intermediate states that seem to be in equilibrium until a ring is captured by GroES65. ATP binding causes small movements of the equatorial domains that are relayed both within and between rings. In the ATP-bound ring, the movements are amplified into large rotations of the apical domains that culminate in dramatic reorganization of the substrate-binding surface. The movements involve a rotation about the equatorial–intermediate hinge, bringing the catalytic Asp residue near the nucleotide-binding pocket. This rotation leads to the breakage of two intersubunit salt bridges and transient generation of two new ones. In addition, the ATP-triggered domain movements are relayed through helix D (FIG. 3b) to the opposite ring via distortion of the inter-ring interface, thus mediating negative cooperativity73.

The recently solved crystal structure of a GroEL double mutant lacking two key salt bridges reveals a remarkable, asymmetric ring with ADP bound to every subunit74. The seven different subunit conformations correspond to those seen in the individual cryo-EM reconstructions65. In the cryo-EM structures, the rings were observed to maintain sevenfold symmetry, except for the apical domains in the more open states.

The observed arrangements of the substrate-binding surface fall into four categories (Supplementary information S1 (figure)). The distal part of the hydrophobic site is delineated by helix H and helix I. The collinear tracks of both helices lining the apo GroEL ring are distorted into tilted tracks with the end of helix H joined to the next helix I in one category of GroEL–ATP states. In these structures, the hydrophobic sites form a continuous band. In the more open GroEL–ATP states, the contacts between adjacent apical domains are completely lost, and the hydrophobic band becomes discontinuous. The free apical domains are not constrained to remain in symmetric positions. The open state has two important properties. First, radial expansion provides a plausible mechanism for forced unfolding of multivalently bound substrate. Second, combined with ring expansion, the elevation of the helix H–helix I groove creates a suitable docking site for the GroES mobile loops. In order to reach the folding-active, GroES-bound conformation, the GroES binding sites must each twist by 100° (Supplementary information S2 (movie)). Thus, the open state is a good candidate for the initial GroES-docked intermediate: the mobile loops are highly flexible and can easily be modelled without the twist they adopt in the GroEL–GroES crystal structures. Moreover, the key parts of the substrate-binding site, helix I and the more proximal, underlying segment, are still exposed to the cavity. It has been proposed that a ternary complex between the open state GroEL–ATP, substrate and GroES represents the elusive intermediate, and that the 100° twist, triggered by binding of the GroES loops to produce the final bullet complex, would provide the power stroke that removes the hydrophobic binding site from the cavity and forcefully ejects the bound substrate into the chamber for folding65.

Group II chaperonins

Group II chaperonins perform similar functions to group I chaperonins, and the underlying machinery is closely related. The most obvious structural difference between group I and group II chaperonins is the presence of a prominent insertion in the apical domain in group I chaperonins, which acts as a substitute for GroES in capping the ring75. The various archaeal forms usually have eightfold or ninefold symmetry, and eukaryotic CCT has eight related but distinct gene products forming the eight subunits of each ring. CCT in particular has been very difficult to study, and even the order of subunits in a ring is controversial76,77. Unlike the archaeal forms and most other chaperones, CCT does not seem to be a HSP. CCT has specialized subunits, with some binding known substrates such as actin and tubulin. Various open, intermediate and closed conformations of intact group II complexes have been described by X-ray crystallography and cryo-EM (for example, REFS 78–81). An interesting difference in how the allosteric machinery operates is that the interring interface is formed of 1:1 instead of 1:2 subunit contacts, leading to altered allosteric interactions82,83. The unliganded form is open and dynamic, equivalent to the open state of GroEL. ATP analogue binding seems to gradually close the cage. Remarkably, although the apical domains undergo similar elevations and twists in group I and II chaperonins, these motions seem to occur in reversed sequence (Supplementary information S3 (movie)). Overall, it seems likely that group II chaperonins perform similar actions as members of the group I family, but they exhibit different ATP-driven allosteric movements.

HSP100 disassembly machines

The HSP100 proteins are unfoldases and disaggregases, forceful unfolding motors that deliver substrates to compartmentalized proteases or disassemble aggregates containing misfolded proteins.

The AAA+ chaperones

HSP100 proteins are members of the AAA+ superfamily, which typically form oligomeric ring structures and have mechanical actions such as threading polypeptides or polynucleotides through a central channel in order to unfold or unwind them84,85. AAA+ proteins function in various cellular processes, including the disassembly of complexes, for example the SNARE complexes that bring membranes together for vesicle fusion. The role of chaperone members of this family is best characterized in regulated proteolysis. At the core of these compartmentalized proteases is a stack of co-axial ATPase and protease rings, formed either by separate functional domains of a single subunit type (as in the bacterial Lon protease) or in separate ATPase and protease subunit rings (as in the HslUV (also known as ClpYQ) complex)86 (FIG. 4a,b). In HslUV, both rings are hexameric, whereas others such as ClpAP have a symmetry mismatch with hexameric ClpA ATPase and heptameric ClpP protease rings87. Although the eukaryotic proteasome is much more complex, it has the same core architecture, and its regulatory cap contains a heterohexamer of ATPase subunits (RPT1–RPT6) that performs the same unfolding and threading functions88,89.

Figure 4. HSP100 unfoldase.

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a ∣ The two types of heat shock protein 100 (HSP100) sequences are shown schematically, with either a single or two tandem AAA+ domains. The characteristic Walker A and B sites are shown in red. b ∣ The HslUV ATPase–protease complex is shown as a cartoon on the left, and the atomic structure is shown on the right (Protein Data Bank (PDB) code: 1G3I)124. c ∣ Top view of the asymmetric ClpX crystal structure (PDB code: 3HWS)95. The four bound ADP molecules are shown in space-filling format and Tyr side chains on the pore loops are shown as magenta sticks. d ∣ Side view section of ClpX showing the pore with three of the Tyr sites at different heights.

The defining feature of the superfamily is the AAA+ domain, which consists of an α–β subdomain and a smaller, helical subdomain85 (FIG. 4c,d). The nucleotide-binding site is located at the subdomain interface. Conserved regions important in nucleotide binding and hydrolysis are the Walker A and Walker B motifs, sensor 1 and sensor 2 as well as the Arg finger involved in catalysis of the ATPase at the interface between subunits. AAA+ chaperones typically form hexameric rings that surround a narrow central pore lined with loops containing a substrate interaction site with aromatic and hydrophobic side chains. They exist as both single AAA+ rings (such as in HslU and ClpX) and stacked rings of tandem AAA+ domains (such as in ClpA, ClpB and ClpC). The HSP100 chaperones also have very mobile N-terminal domains that can play a part in substrate delivery to the central channel or interact with cofactors90,91.

Unfolding during ATP-dependent proteolysis

How does unfolding work? First, the substrate is targeted to the entrance of the HSP100 channel. In bacteria, ribosome stalling causes expressed polypeptides to be marked for degradation by addition of an 11-residue peptide, the small, stable 10S RNA ssrA tag, which targets them to ClpXP or ClpAP92. Both ClpX and ClpA are powerful unfoldases that can even rapidly unfold a stable protein like GFP, if it is suitably tagged93. The central channel is lined with Tyr residues on mobile pore loops that provide the binding sites for translocating chains, without specificity for sequence or chain polarity94 (FIG. 4c,d). Once a polypeptide terminus or loop is engaged in the channel, rotations of the AAA+ subdomains, fuelled by the ATPase cycle, are thought to produce a rowing motion to spool the unfolding chain through the channel. The structure of an asymmetric ClpX ring shows a sequence of pore loops at different heights in the channel and suggests a sequential or random action of the sub units around the ring95 (FIG. 4d). Their axial separation of ~1 nm fits well with results obtained from single-molecule optical tweezer experiments showing translocation steps in multiples of 1 nm96. Force and extension measurements support the action of a power stroke rather than a ratchet mechanism capturing random Brownian motions. The single-molecule approach shows that a C-terminal subdomain of GFP is extracted first, and this destabilizes the rest of the β-barrel, which unfolds before it is delivered to the surface of ClpX. Thus, for GFP, only the first unfolding step requires forceful pulling.

The AAA+ protein p97 (also known as CDC48 or VCP) functions in the transport of substrates to the proteasome, in particular of proteins that are misfolded in the ER and are retrotranslocated to the cytosol for degradation97,98. p97 is a highly conserved protein with tandem AAA+ domains and a mobile N-terminal domain and has recently been suggested to represent the ancestral proteasome unfoldase ring99. p97, together with cofactors, has various other roles when it is in close proximity to membranes. These functions relate more to the actions of family members such as NSF (N-ethylmaleimide-sensitive factor), which disassemble SNARE complexes at membrane surfaces after they have mediated vesicle fusion.

Protein disaggregation

A subset of the HSP100 chaperones found in bacteria, plants and fungi have the unique ability to reverse protein aggregation, in cooperation with their cognate HSP70 system100–102. This subfamily includes E. coli ClpB and yeast Hsp104, which have tandem ATPase domains. A 90 Å long coiled-coil propeller, inserted near the end of the first ATPase domain103, couples their unfolding and translocation actions to HSP70 (REF. 104) (FIG. 5a). Binding of the HSP70 ATPase domain to one end of the coiled-coil, a region highly sensitive to mutations and known as motif 2, is required for disaggregation105,106. Docking to low-resolution and symmetrized electron microscopy maps yielded controversial results regarding the hexamer arrangement and the degree of expansion of the ring. A model based on studies of Thermus thermophilus ClpB proposes that the subunits are tightly packed around a 15 Å channel and the coiled-coils protrude as radial spikes103. By contrast, electron microscopy maps of yeast Hsp104 suggest a much more expanded ring with a wide channel and the coiled-coils intercalated between the subunits, partly buried and partly exposed on the surface, with the HSP70-binding tip of the coil adjacent to the N-terminal ring107. More recent cryo-EM maps of HSP104 are interpreted as typical AAA+ rings with the coiled-coils on the outside, but no density is observed for the coiled-coils108. A low-resolution crystal structure of the hexameric assembly of ClpC, a protease-coupled HSP100 with tandem AAA+ domains and a partial coiled-coil structure, shows an expanded ring and the coiled-coil lying tangentially on the surface109. However, ClpC lacks the HSP70-binding arm of the coil and requires the cofactor MecA for hexamer assembly. Recent work probing accessibility and hydrogen–deuterium exchange on the coiled-coil domain of E. coli ClpB does not support either model. Rather, it suggests that the coil lies on the surface of the hexamer, with motif 2 being protected when ClpB activity is repressed and being accessible when ClpB is active110 (FIG. 5b).

단백질 분해

박테리아, 식물 및 곰팡이에서 발견되는 HSP100 샤페론의 하위 집합은 동족 HSP70 시스템100-102 과 협력하여 단백질 응집을 역전시키는 독특한 능력을 가지고 있습니다. 이 아과에는 대장균 ClpB와 효모 Hsp104가 포함되며, 이들은 ATPase 도메인을 함께 가지고 있습니다. 90Å 길이의 코일 코일 프로펠러는 첫 번째 ATPase 도메인103 의 끝 근처에 삽입되어 전개 및 전위 작용을 HSP70에 결합합니다(참조 104)(그림 5a). 돌연변이에 매우 민감하고 모티프 2로 알려진 영역인 코일 코일의 한쪽 끝에 HSP70 ATPase 도메인을 결합하는 것은 해체에 필요합니다105,106. 저해상도 및 대칭 전자 현미경 맵에 도킹한 결과, 헥사머 배열과 고리의 확장 정도에 대해 논란이 있는 결과가 나왔습니다. 써머스 써모필루스 ClpB에 대한 연구에 기반한 모델은 서브유닛이 15Å 채널 주위에 촘촘히 밀집되어 있고 코일 코일이 방사형 스파이크처럼 돌출되어 있다고 제안합니다103. 이와 대조적으로 효모 Hsp104 의 전자 현미경 지도는 넓은 채널을 가진 훨씬 더 확장된 고리와 서브유닛 사이에 코일 코일이 끼어 있고, 일부는 묻혀 있고 일부는 표면에 노출되어 있으며, 코일의 HSP70 결합 끝이 N-말단 고리에 인접해 있음을 시사합니다107. HSP104의 최근 cryo-EM 지도는 외부에 코일 코일이 있는 전형적인 AAA+ 고리로 해석되지만 코일 코일에는 밀도가 관찰되지 않습니다108. 탠덤 AAA+ 도메인과 부분 코일 코일 구조를 가진 프로테아제 결합 HSP100인 ClpC의 육사메릭 조립체의 저해상도 결정 구조는 확장된 링과 표면에 접선 방향으로 놓인 코일 코일을 보여줍니다109. 그러나 ClpC는 코일의 HSP70 결합 암이 부족하고 헥사머 조립을 위해 보조 인자 MecA가 필요합니다. 대장균 ClpB의 코일-코일 도메인에서 접근성과 수소-중수소 교환을 조사한 최근 연구는 두 모델을 모두 지지하지 않습니다. 오히려 코일은 헥사머 표면에 놓여 있으며, ClpB 활동이 억제되면 모티브 2가 보호되고 ClpB가 활성화되면 접근이 가능하다는 것을 시사합니다110 (그림 5b).

Figure 5. HSP100–HSP70 disaggregase.

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The crystal structure of a ClpB subunit (Protein Data Bank (PDB) code: 1QVR)103 (part a) and a schematic representation of the three-tiered hexamer are shown, with one ClpB coiled-coil domain (dark blue) bound to heat shock protein 70 (HSP70; with the nucleotide-binding domain shown in green and the substrate-binding domain in blue (PDB code: 4B9Q)) (part b). The ClpB–Hsp70 complex is derived from the model in REF. 106 combined with the structure of domain-docked HSP70 from REF. 20. The motif 2 sequence in the coiled-coil domain is highlighted in pink. A substrate polypeptide (yellow) is being extracted from an aggregate and threaded through the ClpB channel.

Methyl TROSY NMR has recently been used to model the local interactions between the ClpB coiled-coil and the DnaK ATPase domain in its open, ADP-bound state106. Combining this model with the model of domain-docked DnaK suggests how DnaK might deliver a polypeptide segment to ClpB (FIG. 5b). This speculative, combined model suggests that the ClpB coiled-coil adopts a more vertical orientation to bring the DnaK substrate-binding domain to the vicinity of the pore channel. The N-terminal domains of ClpB might play a part in delivering the substrate from DnaK to ClpB, after DnaJ makes initial weak contact with the surface of the aggregate and hands over a segment of the polypeptide for engagement with DnaK.

Conclusions

Chaperones are nanoscale molecular machines that recognize incompletely or incorrectly folded proteins, arrest or unfold them and then either release them for spontaneous refolding or target them for degradation. With the help of many cofactors, the general purpose chaperone HSP70, a two-domain monomer, carries out all these actions. Another ‘sociable’ chaperone, HSP90, acts as a flexible dimer, with even more partners to regulate its activities. In a more solitary action, the HSP60 chaperonins assist folding by creating an isolation chamber for the substrate protein. Most forceful of all, the HSP100 protein remodellers can rip apart even stably folded proteins or disassemble large and otherwise irreversible aggregates.

HSP70 and HSP90 have many surface exposed interaction sites for cofactors, giving them a high degree of regulation and integration into other cellular pathways. By contrast, the HSP60 and HSP100 families are largely inward looking, and they enclose their active sites with few cofactors. Their activities are mainly regulated by a stress-induced increase in their expression‎ levels. A striking feature of the ATPase cycles of these chaperones is their highly dynamic nature. Rather than simple conformational switching, the massive domain movements in chaperone action are only loosely coupled to their nucleotide-bound state. Nevertheless, each of these chaperone families has a distinct mode of ATP binding, ranging from the unique chaperonin nucleotide site to the very widespread Walker A and Walker B type ATPase in HSP100. HSP70 shares its nucleotide-binding fold with actin and hexokinase, whereas HSP90 has a GHKL nucleotide-binding fold characteristic of DNA gyrase. The nucleotide binds in an extended conformation to HSP60 and HSP70 but is bent when bound to HSP90 and HSP100, giving rise to different specificities for nucleotide analogues (Supplementary information S4 (figure)).

Although the chaperone systems discussed here have a fairly broad range of substrates, many proteins have specific requirements for chaperones and co-chaperones. For example, the substrates of group I and group II chaperonins are quite distinct; specific HSP40 co-chaperones are required together with HSP70 for the folding of many important substrates. The mechanisms of this specificity are poorly understood. A major current question is why the chaperone systems become less effective in ageing organisms, leading to the eventual failure of protein quality control and the onset of misfolding diseases. Future progress in the field will require high-resolution structures of chaperone complexes acting on misfolded or unfolded proteins, the identification of specific causal pathways in aggregate and amyloid toxicity, as well as a better understanding of the regulation of proteostasis.

결론

샤페론은 불완전하거나 잘못 접힌 단백질을 인식하고, 이를 잡아두거나 펼친 다음 자발적으로 다시 접히도록 풀어주거나 분해를 목표로 삼는 나노 크기의 분자 기계입니다. 많은 보조 인자의 도움으로 두 도메인 단량체인 범용 샤프론 HSP70이 이러한 모든 작용을 수행합니다. 또 다른 '사교적인' 샤프론인 HSP90은 더 많은 파트너와 함께 활동을 조절하는 유연한 이량체 역할을 합니다. 보다 고독한 작용을 하는 HSP60 샤페론은 기질 단백질의 분리 챔버를 만들어 폴딩을 돕습니다. 무엇보다도 가장 강력한 HSP100 단백질 리모델러는 안정적으로 접힌 단백질도 찢어버리거나 돌이킬 수 없는 큰 응집체를 분해할 수 있습니다.

HSP70과 HSP90은 보조 인자와의 상호작용 부위가 표면에 많이 노출되어 있어 다른 세포 경로에 높은 수준의 조절과 통합을 제공합니다. 반면, HSP60 및 HSP100 계열은 대부분 내부에 위치하며 보조 인자가 거의 없는 활성 부위를 둘러싸고 있습니다. 이들의 활동은 주로 스트레스로 인한 발현 수준 증가에 의해 조절됩니다. 이러한 샤프론의 ATPase 주기의 두드러진 특징은 매우 역동적이라는 점입니다. 단순한 형태 전환이 아니라, 샤페론 작용의 대규모 도메인 이동은 뉴클레오티드 결합 상태와 느슨하게 결합되어 있습니다. 그럼에도 불구하고 이러한 각 샤페론 계열은 고유한 샤페론인 뉴클레오티드 부위부터 HSP100의 매우 광범위한 워커 A 및 워커 B 유형 ATPase에 이르기까지 각기 다른 ATP 결합 방식을 가지고 있습니다. HSP70은 액틴 및 헥소키나아제와 뉴클레오타이드 결합 폴드를 공유하는 반면, HSP90은 DNA 자이라아제의 특징인 GHKL 뉴클레오타이드 결합 폴드를 가지고 있습니다. 뉴클레오타이드는 HSP60 및 HSP70에는 확장된 형태로 결합하지만 HSP90 및 HSP100에 결합하면 구부러져 뉴클레오티드 유사체에 대해 다른 특이성을 나타냅니다(보충 정보 S4(그림)).

여기서 설명하는 샤페론 시스템은 상당히 광범위한 기질을 가지고 있지만, 많은 단백질은 샤페론과 공동 샤페론에 대한 특정 요구 사항을 가지고 있습니다. 예를 들어, 그룹 I과 그룹 II 샤페론의 기질은 상당히 구별되며, 많은 중요한 기질을 접으려면 HSP70과 함께 특정 HSP40 코-샤페론이 필요합니다. 이러한 특이성의 메커니즘은 잘 알려져 있지 않습니다. 현재 주요한 의문은 왜 노화된 유기체에서 샤프론 시스템의 효율성이 떨어지고 결국 단백질 품질 관리의 실패와 잘못된 폴딩 질환의 발병으로 이어지는가 하는 것입니다. 이 분야의 향후 진전을 위해서는 잘못 접히거나 펼쳐진 단백질에 작용하는 샤프론 복합체의 고해상도 구조, 응집 및 아밀로이드 독성의 특정 인과 경로의 규명, 그리고 단백질 정체 조절에 대한 더 나은 이해가 필요할 것입니다.

Supplementary Material

Movie 1

Download video file (4.8MB, mov)

Movie 2

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SFig 1

NIHMS61973-supplement-SFig_1.pdf (454.1KB, pdf)

SFig 4

NIHMS61973-supplement-SFig_4.pdf (360.5KB, pdf)

Acknowledgements

The author is grateful to D. Clare, C. Vaughan, J. Trapani and D. Middendorf for helpful comments on the manuscript and thanks the Wellcome Trust for funding.

GlossaryAutophagy

A process in which intracellular material is enclosed in a membrane compartment and delivered to the lysosome (vacuole in yeast) for degradation and recycling of the macromolecular constituents.

Unfolded protein response

(UPR). A signalling system that regulates the balance between folding capacity of the endoplasmic reticulum (ER) and protein synthesis. If misfolded proteins accumulate, this pathway triggers apoptosis.

Heat shock proteins

(HSPs). The expression‎ of these proteins is greatly enhanced by increased temperature or other stress conditions. Most chaperones are HSPs.

Allosteric machines

Macromolecular complexes in which the activity is indirectly modulated by binding of an effector at a site remote from the active site. This induces shifts in the domain or subunit structure that influence the conformation of the active site.

Amyloid

Protein species that form deposits consisting of fibrillar protein aggregates rich in β-sheet structure. They assemble from proteins that have unfolded or misfolded. About 20 distinct protein species are associated with particular amyloid diseases.

Methyl transverse relaxation optimized spectroscopy

(methyl TROSY). A method that uses selective isotope labelling of methyl groups on protein side chains with a transverse relaxation scheme optimized for methyl groups to obtain well-resolved nuclear magnetic resonance (NMR) spectra from large protein structures far beyond the normal range obtained in NMR structure determination.

GHKL

An ATP-binding superfamily that includes DNA gyrase, the molecular chaperone heat shock protein 90, the DNA-mismatch-repair enzyme MutL and His kinase, which bind ATP in a characteristic bent conformation.

Footnotes

Competing interests statement The author declares no competing financial interests.

SUPPLEMENTARY INFORMATION See online article: S1 (table) ∣ S2 (movie) ∣ S3 (movie) ∣ S4 (figure)

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