A dynamics based analysis of allosteric modulation in heat shock proteins

The 70 kDa and 90 kDa heat shock proteins (Hsp70 and Hsp90) are molecular chaperones that play central roles in maintaining cellular homeostasis in all organisms of life with the exception of archaea. In addition to their general chaperone function in protein quality control, Hsp70 and Hsp90 cooperate in the regulation and activity of some 200 known natively folded protein clients which include protein kinases, transcription factors and receptors, many of which are implicated as key regulators of essential signal transduction pathways. Both chaperones are considered to be large multi-domain proteins that rely on ATPase activity and co-chaperone interactions to regulate their conformational cycles for peptide binding and release. The unique positioning of Hsp90 at the crossroads of several fundamental cellular pathways coupled with its known association with diverse oncogenic peptide clients has brought the molecular chaperone under increasing interest as a potential anti-cancer target that is crucially implicated with all eight hallmarks of the disease. Current orthosteric drug discovery efforts aimed at the inhibition of the ATPase domain of Hsp90 have been limited due to high levels of associated toxicity. In an effort to circumnavigate this, the combined focus of research efforts is shifting toward alternative approaches such as interference with co-chaperone binding and the allosteric inhibition/activation of the molecular chaperone. The overriding aim of this thesis was to demonstrate how the computational technique of Perturbation response scanning (PRS) coupled with all-atom molecular dynamics simulations (MD) and dynamic residue interaction network (DRN) analysis can be used as a viable strategy to efficiently scan and accurately identify allosteric control element capable of modulating the functional dynamics of a protein. In pursuit of this goal, this thesis also contributes to the current understanding of the nucleotide dependent allosteric mechanisms at play in cellular functionality of both Hsp70 and Hsp90. All-atom MD simulations of E. coli DnaK provided evidence of nucleotide driven modulation of conformational dynamics in both the catalytically active and inactive states. PRS analysis employed on these trajectories demonstrated sensitivity toward bound nucleotide and peptide substrate, and provided evidence of a putative allosterically active intermediate state between the ATPase active and inactive conformational states. Simultaneous binding of ATP and peptide substrate was found to allosterically prime the chaperone for interstate conversion regardless of the transition direction. Detailed analysis of these allosterically primed states revealed select residue sites capable of selecting a coordinate shift towards the opposite conformational state. In an effort to validate these results, the predicted allosteric hot spot sites were cross-validated with known experimental works and found to overlap with functional sites implicated in allosteric signal propagation and ATPase activation in Hsp70. This study presented for the first time, the application of PRS as a suitable diagnostic tool for the elucidation and quantification of the allosteric potential of select residues to effect functionally relevant global conformational rearrangements. The PRS methodology described in this study was packaged within the Python programming environment in the MD-TASK software suite for command-line ease of use and made freely available. Homology modelling techniques were used to address the lack of experimental structural data for the human cytosolic isoform of Hsp90 and for the first time provided accurate full-length structural models of human Hsp90α in fully-closed and partially-open conformations. Long-range all-atom MD simulations of these structures revealed nucleotide driven modulation of conformational dynamics in Hsp90. Subsequent DRN and PRS analysis of these MD trajectories allowed for the quantification and elucidation of nucleotide driven allosteric modulation in the molecular chaperone. A detailed PRS analysis revealed allosteric inter-domain coupling between the extreme terminals of the chaperone in response to external force perturbations at either domain. Furthermore PRS also identified several individual residue sites that are capable of selecting conformational rearrangements towards functionally relevant states which may be considered to be putative allosteric target sites for future drug discovery efforts Molecular docking techniques were employed to investigate the modulation of conformational dynamics of human Hsp90α in response to ligand binding interactions at two identified allosteric sites at the C-terminal. High throughput screening of a small library of natural compounds indigenous to South Africa revealed three hit compounds at these sites: Cephalostatin 17, 20(29)-Lupene-3β isoferulate and 3'-Bromorubrolide F. All-atom MD simulations on these protein-ligand complexes coupled with DRN analysis and several advanced trajectory based analysis techniques provided evidence of selective allosteric modulation of Hsp90α conformational dynamics in response to the identity and location of the bound ligands. Ligands bound at the four-helix bundle presented as putative allosteric inhibitors of Hsp90α, driving conformational dynamics in favour of dimer opening and possibly dimer separation. Meanwhile, ligand interactions at an adjacent sub-pocket located near the interface between the middle and C-terminal domains demonstrated allosteric activation of the chaperone, modulating conformational dynamics in favour of the fully-closed catalytically active conformational state. Taken together, the data presented in this thesis contributes to the understanding of allosteric modulation of conformational dynamics in Hsp70 and Hsp90, and provides a suitable platform for future biochemical and drug discovery studies. Furthermore, the molecular docking and computational identification of allosteric compounds with suitable binding affinity for allosteric sites at the CTD of human Hsp90α provide for the first time “proof-of-principle” for the use of PRS in conjunction with MD simulations and DRN analysis as a suitable method for the rapid identification of allosteric sites in proteins that can be probed by small molecule interaction. The data presented in this section could pave the way for future allosteric drug discovery studies for the treatment of Hsp90 associated pathologies

Molecular Dynamics Simulation Analysis Of His226 Mutation On The Dynamics Of The Atpase Domain Of Dnak

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2017Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2017Proteinler, canlılar için yaşamsal açıdan önemli makromoleküllerdir. Sentezlendikten sonra fonksiyonel özelliklerini kazanmaları içinse doğru şekilde katlanmaları şarttır. Peptid zinciri sentezlenirken, üç boyutlu yapısını kazanması sürecinde görülen ilk etkileşimler hidrofobik etkileşimlerdir. Sonrasında ise iyonik etkileşimler,van der Waals etkileşimleri, dipol-dipol etkileşimleri, hidrojen bağları ile birlikte peptit zincirleri fonksiyonel hale geldiği üç boyutlu yapısına ulaşır. Bazı küçük proteinler bu şekilde tek başına katlanabilirken, pek çok protein katlanabilmek için şaperonlara ihtiyaç duyar. Proteinlerin düzgün katlanamaması ise, Alzheimer, Parkinson gibi çeşitli nörodejeneratif hastalıklara sebebiyet verebilir. Şaperonlar ilk kez 1978 yılında Ron Laskey tarafından bulundu. 1987 yılında R. John Ellis tarafından yapılan çalışmalarla birlikte, bu konudaki araştırmalar hız kazandı. Eksikliklerinde nörodejeneratif hastalıkların oluştuğunun keşfedilmesi ile beraber, şaperonlar ile ilgili çalışmalar oldukça sık gündeme gelmeye başlamıştır. Şaperonlar, normal şartlarda hücrede normal seviyelerde sentezlenir. Stres koşulları ise bu durumu değiştirebilir. Şaperonlar hücrenin aşırı ısı ve pH değişiminden olumsuz etkilenmemesini sağlamanın yanında oksidatif stres ve kimyasal stres gibi durumlara karşı da hücreyi korur. Bu gibi streslere karşı hücrenin bir savunma stratejisi olarak, şaperonların ekspresyonları artar ve tehdit altında olan hücre içi yaşamsal öneme sahip enzimlerin ve diğer proteinlerin yapısının bozularak fonksiyonel olarak zarar görmeleri engellenir. Hsp70, şaperon ailesinin en bilinen ve en yaygın üyesidir. Şaperonlar evrimsel süreçte korunmuş moleküler şaperonlar arasında yer almaktadır. Bakteri, arkea ve ökaryotlarda olmak üzere neredeyse tüm hücrelerde bulunurlar ve hayati açıdan önemli rollere sahiptirler. Örneğin, yeni eksprese edilmiş peptit zincirlerinin katlanması, agregat oluşumunun önlenmesi ya da proteinlerin belirli organellere translokasyonu Hsp70 tarafından gerçekleştirilir. Hsp70, katlanacak proteinin hidrofobik bölgelerine bağlanarak bu bölgelerin birbirleriyle doğru olmayan şekilde etkileşmesini önler, bu sayede proteinin yanlış katlanmasını engeller. Bu sebepten ötürü, Hsp70'in proteinlerin katlanmasında katalizör görevi görmediği, katlanması gereken proteinler için uygun ortam oluşturduğu düşünülmektedir. Çalışmamızda kullanılan E.coli Hsp70 homoloğu olan DnaK iki domenden oluşmaktadır. Bunlardan biri amino (N) ucunda bulunan nükleotit bağlayan ve ATPaz aktivitesi olan 44 kDa'lık Nükleotit Bağlanan Domen (NBD), diğeri ise karboksil (C) ucunda bulunan ve substrat bağlayan 25 kDa'lık Subsrat Bağlanan Domen (SBD)'dir. Bu iki domen ise oldukça korunmuş, domenler arasında bulunan xxiv hidrofobik bir bağlaç ile bağlanır. NBD ve SBD arasında allosterik bir etkileşim mevcuttur. Bu iki domen arasındaki iletişim ise domenler arasındaki bağlaç tarafından sağlanmaktadır. Her iki domende de gerçekleşen konformasyonel değişiklikler sonucunda, ATP bağlanması ve hidrolizi substrat affinitesini düzenlerken, ATP hidrolizi ise substrat bağlanması ile tetiklenmektedir. Yapılan kristalografi ve NMR çalışmaları sonucunda elde edilen yapılarda, ADP bağlı halde, bu iki domen ve bağlaç birbirinden ayrı şekilde gözlemlenmektedir. Bu durumda SBD'nin substrata olan afinitesi yüksektir ve dolayısıyla SBD, katlanacak protein üzerine kapanmış durumdadır. NBD'ye ATP bağlandığında ise, SBD açık bir konformasyonda ve bağlacı NBD'nin içlerine alacak şekilde NBD ile etkileşmektedir. Bu konformasyonda SBD'nin substrata afinitesi düşüktür ve katlanmış proteinin salınması ATP bağlı halde gerçekleştirilir. Sonrasında, SBD'ye bağlanan katlanması gerekli yeni bir protein, NBD'ye bağlı halde bululanan ATP'nin hidrolizini tetikler. ATP'nin ADP'ye hidrolizi sırasında SBD katlanması gereken proteinin üzerine kapanır, NBD ve SBD birbirinden tekrar ayrılır ve mekanizma bu döngü ile devam eder. Bu döngüye koşaperonlar eşlik eder. Hsp40 ailesi ve nükleotit değişim ailesi bu döngüde görevli koşaperonlar olarak bilinir. Hsp40 ailesi (DnaK için DnaJ), ATP hidrolizini hızlandırmakla görevliyken, nükleotit değişim faktörleri (DnaK için GrpE) ise hidroliz ile oluşan nükleotiti değiştirmek ve Hsp70'yi yeni döngüye hazırlama görevindedir. Mayer ve Bukau'nun 2015 yılında yaptığı son çalışmalar, ATP ile indüklenen substrat salınımının, substratın bağlanmasıyla uyarılan ATP hidrolizine göre şaperonun aktivitesinde daha önemli bir rol oynadığını ortaya koymaktadır. 2007 yılında, Swain grubu tarafından yapılan çalışmalarla, domenler arası bağlacın hidrofobik 389VLLL 392 sekansının NBD ve SBD arasındaki allosterik ilişkiden sorumlu olduğu bulunmuştur. Yine bu çalışmaya göre, bağlaç varlığında DnaK (1- 392), substrat tarafından uyarılmış yabanıl tip DnaK gibi davranmaktadır. Yabanıl tip, substrat ile uyarılmamış DnaK'nin aksine, DnaK (1-392)'nin pH bağımlı ve daha yüksek ATPase aktivitesi vardır. Diğer bir taraftan ise, bağlaç yokluğunda DnaK (1- 388), substrat tarafından uyarılmamış yabanıl tip DnaK'yi taklit etmektedir. Moleküler dinamik simulasyonları, 1950 yıllarının ikinci yarısına doğru ortaya çıkmıştır. 1960'larda daha da gelişmiş ve ilk kez 1977 yılında bovin pankreatik tripsin inibitörü ile birlikte proteinler üzerinde kullanılmaya başlanmıştır. In vivo ve in vitro olarak gözlemlemekte güç olabilecek bazı konularda fikir vermek için kullanılan önemli yollardan biri haline gelmiştir. Biyokimya ve biyofizikte oldukça geniş bir uygulama alanı bulmuştur. Bu çalışma DnaK (1-392) ve (1-388) kesik yapılarında, ATP bağlı durumda konformasyonel ve dinamiksel farklılıkları göstermeyi amaçlamanın yanısıra 226. konumdaki histidinin alanine mutasyonu halinde genel konformasyon ise de nasıl bir değişim olduğunu moleküler dinamik analizleriyle göstermeyi amaçlamaktadır. Kesik yapılar 4JN4 kodlu PDB (protein data bank) dosyasından modifiye edilip, NAMD/CHARMM-GUİ kullanılarak 200 ns boyunca yürütülmüştür. Bu süreçte ortalama karekökten sapma (RMSD) ve ortalama karekök değişimi (RMSF) gibi temel moleküler dinamik analizlerinin yanısıra, Başlıca Komponent Analizi (PCA), hedef amino asitler için zamana bağımlı uzaklık ölçümleri ve proteinin ne kadar kompakt olduğunu ölçmek amacıyla dönüş çapı (Rg) analizi yapılmıştır. Yapılan analizler sonucu bağlacın 389VLLL392 kısmının bulunmadığı DnaK (1-388) kesik yapısının, bağlacın bulunduğu DnaK (1-392) yapısına gore belirli bölgelerde xxv daha kapalı bir konformasyonda olduğu gözlemlenmiştir. Ayrıca bu iki yapı arasında farklı dinamikler olduğu da görülmektedir. Bir diğer önemli sonuç ise bağlaç varlığında His226'nın bu dinamikte önemli bir rolü olduğu yönündedir. 226 numaralı Histidinin alanine mutasyonu ile birlikte, konformasyonun daha da açıldığı, bazı amino asitlerin de bulunduğu konumdan oldukça saptığı görülmüştür. Bunlardan önemli olarak gördüğümüz katalitik bölgede bulunan ve ATP hidrolizinden sonra fosfatı kabul eden amino asit olduğu düşünülen 199. konumdaki Treonin yakından incelendiğinde simulasyon sürecinin sonlarına doğru ATP'den aniden ve önemli ölçüde uzaklaştığı görülmüştür. Bu durumun doğurabileceği sonuçlar, araştırma grubumuza ait diğer deneysel verileri desteklemekle birlikte Histidinin Hsp70 genel dinamiği için ne kadar önemli olduğunu da göstermektedir. Uzun vadede, yapılan bu moleküler dinamik simulasyon çalışmalarının Hsp70 çalışma mekanizmasını detaylı bir şekilde ortaya koyarak Hsp70 kaynaklı nörodejenaratif hastalıkların tedavisine ışık tutabileceği düşünülmektedir.Hsp70s are evolutionarily highly conserved ATP-dependent molecular chaperones which are ubiquitously expressed in the cell. They are found in three domains of life and have essential roles in cells, such as aiding proper folding of nascent polypeptides, prevention of polypeptide chains from misfolding and translocation of proteins across membranes. The diverse cellular functions of Hsp70s are based on the recognition of hydrophobic sequences of client protein. Hsp70s corporate with other co-chaperones like nucleotide exchange factors and J-domain proteins. In our study, we used DnaK which is an Escherichia coli homolog of Hsp70. DnaK have an N-terminal nucleotide-binding ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). These two domains are connected by a highly conserved hydrophobic interdomain linker. There is an allosteric communication between the domains via the hydrophobic linker. Substrate affinity is regulated by ATP binding and hydrolysis, which results in conformational changes in both domains, while ATP hydrolysis is stimulated by substrate binding. In 2007, Swain et al. revealed that the conserved hydrophobic 389VLLL392 sequence of the interdomain linker is responsible for the allosteric communication between NBD and SBD. According to this study, DnaK (1-392) behaves like the substrate-stimulated DnaK that is pH-dependent, and shows higher activity than that of the unstimulated fulllength protein. In contrast, DnaK (1-388) mimics the activity of the substrate-free form of the full-length DnaK. Which amino acids in the catalytic site are responsible in allosteric communication and pH-dependent ATPase activity in the presence of linker are not enlightened so far, however there are several research trying to find out the key residues and reveal the detailed mechanism of DnaK. In this study, the effect of linker 389VLLL392 on the ATP-bound protein conformation and H226A mutation on ATP-bound DnaK's (1-392) construct were investigated by using molecular dynamics simulations. MD simulation trajectories were analyzed by root mean square deviation (RMSD) and, root mean square fluctuation (RMSF) analysis, also by distance measurement in a time-dependent manner and, principle component analysis. From these analysis it was found that distance between the helices which contain His226 and its neighbour helix is closer to each other in DnaK (1-388) constructs. Moreover, this study reveals that His226 contributes the stabilization of residue Thr199 which is suspicious as a phosphate acceptor after the hydrolysis of ATP.Yüksek LisansM.Sc

Targeting allosteric sites of Escherichia coli heat shock protein 70 for antibiotic development

Hsp70s are members of the heat shock proteins family with a molecular weight of 70-kDa and are the most abundant group in bacterial and eukaryotic systems, hence the most extensively studied ones. These proteins are molecular chaperones that play a significant role in protein homeostasis by facilitating appropriate folding of proteins, preventing proteins from aggregating and misfolding. They are also involved in translocation of proteins into subcellular compartments and protection of cells against stress. Stress caused by environmental or biological factors affects the functionality of the cell. In response to these stressful conditions, up-regulation of Hsp70s ensures that the cells are protected by balancing out unfolded proteins giving them ample time to repair denatured proteins. Hsp70s is connected to numerous illnesses such as autoimmune and neurodegenerative diseases, bacterial infection, cancer, malaria, and obesity. The multi-functional nature of Hsp70s predisposes them as promising therapeutic targets. Hsp70s play vital roles in various cell developments, and survival pathways, therefore targeting this protein will provide a new avenue towards the discovery of active therapeutic agents for the treatment of a wide range of diseases. Allosteric sites of these proteins in its multi-conformational states have not been explored for inhibitory properties hence the aim of this study. This study aims at identifying allosteric sites that inhibit the ATPase and substrate binding activities using computational approaches. Using E. coli as a model organism, molecular docking for high throughput virtual screening was carried out using 623 compounds from the South African Natural Compounds Database (SANCDB; https://sancdb.rubi.ru.ac.za/) against identified allosteric sites. Ligands with the highest binding affinity (good binders) interacting with critical allosteric residues that are druggable were identified. Molecular dynamics (MD) simulation was also performed on the identified hits to assess for protein-inhibitor complex stability. Finally, principal component analysis (PCA) was performed to understand the structural dynamics of the ligand-free and ligand-bound structures during MD simulation

Hsp70 Phosphorylation: A Case Study of Serine Residues 385 and 400

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, remains elusive. To investigate the impact of PTMs on chaperone function, we chose to characterize two sites of phosphorylation on the linker of HspA1, the stress inducible human Hsp70. To mimic these phosphoserines, we used aspartate as a phosphomimetic substitution for all experiments. Interdomain allostery ties together chaperone structure and function. Therefore, the impact of phosphorylation on interdomain allostery is probed using biophysical and biochemical techniques. Altogether, data suggest that phosphorylation of the linker and SBD destabilizes the chaperone, while shifting the population towards the docked state. This result alludes to a previously described region of the protein that uncouples domain docking from conformational changes in the substrate-binding domain. The cross-communication between these phosphorylation sites reveals a novel, synergistic effect on chaperone structure and function

Allo-network drugs: Extension of the allosteric drug concept to protein-protein interaction and signaling networks

Allosteric drugs are usually more specific and have fewer side effects than orthosteric drugs targeting the same protein. Here, we overview the current knowledge on allosteric signal transmission from the network point of view, and show that most intra-protein conformational changes may be dynamically transmitted across protein-protein interaction and signaling networks of the cell. Allo-network drugs influence the pharmacological target protein indirectly using specific inter-protein network pathways. We show that allo-network drugs may have a higher efficiency to change the networks of human cells than those of other organisms, and can be designed to have specific effects on cells in a diseased state. Finally, we summarize possible methods to identify allo-network drug targets and sites, which may develop to a promising new area of systems-based drug design

Allosteric Regulation at the Crossroads of New Technologies: Multiscale Modeling, Networks, and Machine Learning

Allosteric regulation is a common mechanism employed by complex biomolecular systems for regulation of activity and adaptability in the cellular environment, serving as an effective molecular tool for cellular communication. As an intrinsic but elusive property, allostery is a ubiquitous phenomenon where binding or disturbing of a distal site in a protein can functionally control its activity and is considered as the “second secret of life.” The fundamental biological importance and complexity of these processes require a multi-faceted platform of synergistically integrated approaches for prediction and characterization of allosteric functional states, atomistic reconstruction of allosteric regulatory mechanisms and discovery of allosteric modulators. The unifying theme and overarching goal of allosteric regulation studies in recent years have been integration between emerging experiment and computational approaches and technologies to advance quantitative characterization of allosteric mechanisms in proteins. Despite significant advances, the quantitative characterization and reliable prediction of functional allosteric states, interactions, and mechanisms continue to present highly challenging problems in the field. In this review, we discuss simulation-based multiscale approaches, experiment-informed Markovian models, and network modeling of allostery and information-theoretical approaches that can describe the thermodynamics and hierarchy allosteric states and the molecular basis of allosteric mechanisms. The wealth of structural and functional information along with diversity and complexity of allosteric mechanisms in therapeutically important protein families have provided a well-suited platform for development of data-driven research strategies. Data-centric integration of chemistry, biology and computer science using artificial intelligence technologies has gained a significant momentum and at the forefront of many cross-disciplinary efforts. We discuss new developments in the machine learning field and the emergence of deep learning and deep reinforcement learning applications in modeling of molecular mechanisms and allosteric proteins. The experiment-guided integrated approaches empowered by recent advances in multiscale modeling, network science, and machine learning can lead to more reliable prediction of allosteric regulatory mechanisms and discovery of allosteric modulators for therapeutically important protein targets

Dissecting Structure-Encoded Determinants of Allosteric Cross-Talk between Post-Translational Modification Sites in the Hsp90 Chaperones

Post-translational modifications (PTMs) represent an important regulatory instrument that modulates structure, dynamics and function of proteins. The large number of PTM sites in the Hsp90 proteins that are scattered throughout different domains indicated that synchronization of multiple PTMs through a combinatorial code can be invoked as an important mechanism to orchestrate diverse chaperone functions and recognize multiple client proteins. In this study, we have combined structural and coevolutionary analysis with molecular simulations and perturbation response scanning analysis of the Hsp90 structures to characterize functional role of PTM sites in allosteric regulation. The results reveal a small group of conserved PTMs that act as global mediators of collective dynamics and allosteric communications in the Hsp90 structures, while the majority of flexible PTM sites serve as sensors and carriers of the allosteric structural changes. This study provides a comprehensive structural, dynamic and network analysis of PTM sites across Hsp90 proteins, identifying specific role of regulatory PTM hotspots in the allosteric mechanism of the Hsp90 cycle. We argue that plasticity of a combinatorial PTM code in the Hsp90 may be enacted through allosteric coupling between effector and sensor PTM residues, which would allow for timely response to structural requirements of multiple modified enzymes

Two polymorphisms facilitate differences in plasticity between two chicken major histocompatibility complex class I proteins

Major histocompatibility complex class I molecules (MHC I) present peptides to cytotoxic T-cells at the surface of almost all nucleated cells. The function of MHC I molecules is to select high affinity peptides from a large intracellular pool and they are assisted in this process by co-factor molecules, notably tapasin. In contrast to mammals, MHC homozygous chickens express a single MHC I gene locus, termed BF2, which is hypothesised to have co-evolved with the highly polymorphic tapasin within stable haplotypes. The BF2 molecules of the B15 and B19 haplotypes have recently been shown to differ in their interactions with tapasin and in their peptide selection properties. This study investigated whether these observations might be explained by differences in the protein plasticity that is encoded into the MHC I structure by primary sequence polymorphisms. Furthermore, we aimed to demonstrate the utility of a complimentary modelling approach to the understanding of complex experimental data. Combining mechanistic molecular dynamics simulations and the primary sequence based technique of statistical coupling analysis, we show how two of the eight polymorphisms between BF2*15:01 and BF2*19:01 facilitate differences in plasticity. We show that BF2*15:01 is intrinsically more plastic than BF2*19:01, exploring more conformations in the absence of peptide. We identify a protein sector of contiguous residues connecting the membrane bound ?3 domain and the heavy chain peptide binding site. This sector contains two of the eight polymorphic residues. One is residue 22 in the peptide binding domain and the other 220 is in the ?3 domain, a putative tapasin binding site. These observations are in correspondence with the experimentally observed functional differences of these molecules and suggest a mechanism for how modulation of MHC I plasticity by tapasin catalyses peptide selection allosterically

The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the β6 and β7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the β6/β7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes