Role of Non-Native Electrostatic Interactions in the Coupled Folding and Binding of PUMA with Mcl-1

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye-Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ion strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies.This work was supported by grants to JW from the National Science Foundation (NSF-MCB-0947767 and NSF-PHY-76066, website: www.nsf.gov/), the National Natural Science Foundation of China (91430217, website: www.nsfc.gov.cn/publish/portal1/), and Ministry of Science and Technology of China (2016YFA0203200 and 2013YQ170585, website: http://www.most.gov.cn/eng/); WTC from the National Natural Science Foundation of China (21603217, website: www.nsfc.gov.cn/publish/portal1/), and China Postdoctoral Science Foundation (2016M590268, website: jj.chinapostdoctor.org.cn/V1/Program3/Default.aspx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Probing Order within Intrinsically Disordered Proteins

Decades have passed since the realisation that a protein’s amino acid sequence can contain all the information required to form a complex three-dimensional fold. Until recently, these encoded structures were thought to be crucial determinants of protein function. Much effort was directed to fully understand the mechanisms behind how and why proteins fold, with natively unfolded proteins thought to be experimental artefacts. Today, the field of natively unfolded – or so-called intrinsically disordered – proteins, is rapidly developing. Protein disorder content has been positively correlated with organismal complexity, with over thirty percent of eukaryotic proteins predicted to contain disordered regions. However, the biophysical consequences of disorder are yet to be fully determined. With the aim of addressing some of the outstanding questions, the work described in this thesis focuses on the relevance of structure within disordered proteins. Whilst populating a variety of conformations in isolation, a subset of disordered proteins can fold upon binding to a partner macromolecule. This folded state may be present within the ensemble of conformations sampled by the unbound protein, opening the question of what comes first: folding or binding? Protein engineering techniques were employed to alter the level of residual ‘bound-like’ structure within the free conformational ensemble, and the consequences on coupled folding and binding reactions were investigated. Resultant changes in the rate of association are easily imaginable; yet, this work demonstrates that the majority of the observed changes in binding affinity were due to alterations in the rate of dissociation, thus altering the lifetime of the bound complex. Promiscuous binding is a touted advantage of being disordered. If many disordered proteins, each with their own conformational ensemble, can bind and fold to the same partner, then where is the folding component encoded? Does the partner protein template the folding reaction? Or, is the folding information contained within the disordered protein sequence? Utilising phi-value analysis on the BCL-2 family of proteins, residues in the disordered sequence were probed to ascertain which form contacts at the transition state of the reaction. Comparison with phi-value analyses of alternative pairs – sharing either the ordered or disordered protein – provides insight into the encoding of these interactions. In the context of a bimolecular reaction, the amino acid sequence of the disordered protein was shown to determine the interactions within the transition state. Thus, analogous to the discovery from decades’ past, it is the sequence of the protein that folds which encodes its pathway, even when binding is a prerequisite.BBSRC DTP Studentshi

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies

Intrinsically disordered proteins (IDPs) are characterised by a lack of defined structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. While most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signalling event, are needed to determine mechanisms.This work was supported by the Wellcome Trust (WT 095195MA). M.D.C. is supported by a BBSRC studentship; L.D. by an EPSRC studentship B.I.M.W. by the Cambridge Trust. JC is a Senior Wellcome Trust Research Fellow.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.R115.69271

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions. Here we review and highlight the type of information that can be gained from kinetic studies. In particular, we show how kinetic studies of coupled folding and binding reactions, an important class of signaling event, are needed to determine mechanisms.This work was supported by the Wellcome Trust (WT 095195MA). M.D.C. is supported by a BBSRC studentship; L.D. by an EPSRC studentship B.I.M.W. by the Cambridge Trust. JC is a Senior Wellcome Trust Research Fellow.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.R115.69271

Intrinsically disordered energy landscapes.

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such 'intrinsically disordered' landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an α-helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium.The authors thank Prof. Jane Clarke, Dr. Chris Whittleston, Dr. Joanne Carr, Dr. Iskra Staneva and Dr. David de Sancho for helpful discussions. Y.C. and A.J.B. acknowledge funding from the EPSRC grant number EP/I001352/1, D.C. gratefully acknowledges the Cambridge Commonwealth European and International Trust for financial support and D.J.W. the ERC for an Advanced Grant.This is the final version. It was first published by NPG at http://www.nature.com/srep/2015/150522/srep10386/full/srep10386.html?WT.ec_id=SREP-639%2C638-20150526#abstract

Functional Relevance of Protein Disorder: Why is Disorder Favourable?

For half a century, the central tenet of protein science has been grounded on the idea that the three-dimensional structure of a protein underlies its function. However, increasing evidence of natively unstructured but functional proteins is accumulating. Termed as intrinsically disordered proteins (IDPs), they populate a number of different conformations in isolation. Interestingly, as part of their function, some IDPs become fully or partly structured upon interaction with their binding partners. This process, known as coupled folding and binding raises the question what comes first – folding of the IDP or binding to its partner protein followed by folding. This thesis focuses on understanding the role of disorder in protein- protein interactions using biophysical characterization. Over-representation of IDPs in complex network and signalling pathways emphasizes the importance of disorder. Conformational flexibility in IDPs facilitates post-translational modifications, which provides a neat way to modulate the residual structure. This can alter affinity of IDPs to their partners and it is speculated that bound like structures of IDPs speed association. The impact of phosphorylation was explored in the KID/KIX system: phosphorylation modulates only the dissociation kinetics increasing the lifetime of the bound complex, which may be important in signalling processes. Further, phi-value analysis applied to investigate the mechanism of interaction reveals that non-native interactions play a key role in this reaction, before the IDP consolidates its final structure in the bound complex. Promiscuous interaction of IDPs with their partners often results in complexes with differing affinities. Members of BCL-2 family were explored, and the results indicate that IDPs bind to the same partner protein with marginal variation in the association rates, but significant differences in dissociation rates are observed. Thus, it seems that in such homologous but competing network of proteins, disorder facilitates complexes with differing affinities by modulating dissociation rate, again altering the lifetime of the bound complex. The work presented here demonstrates that disorder plays a role in altering complex lifetimes. Perhaps being disordered permits a level of plasticity to IDPs to adapt the rates at which they bind/unbind to many target proteins. This may be why disorder is conserved and abundant in proteins involved in intricate signalling networks.EPSR

MCL-1 promiscuity and the structural resilience of its binding partners

MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial $\alpha$-helical unfolding in all cases, albeit on strongly varying timescales (1.6~ns for PUMA, 9.7~ns for the previously studied BIM, and 85~ns for NOXA). These differences are interpreted as a BH3-only-specific "structural resilience" to defy the perturbation while remaining in MCL-1's binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1 in general, and the role of the proteins in the apoptotic network

Multiscale Simulations of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack stable secondary and/or tertiary structures under physiological conditions. The have now been recognized to play important roles in numerous biological processes, particularly cellular signaling and regulation. Mutation of IDPs are frequently associated with human diseases, such as cancers and neuron degenerative diseases. Therefore, it is important to understand the structure, dynamics, and interactions of IDPs, so as to establish the mechanistic basis of how intrinsic disorder mediates versatile functions and how such mechanisms may fail in human diseases. However, the heterogeneous structural ensembles of IDPs are not amenable to high resolution characterization solely through experimental measurements, and molecular modelling and simulation are required to study IDP structures, dynamics, and interactions at the atomistic levels. Here, we first applied the state-of-the-art explicit solvent atomistic simulations to an anti-apoptotic protein Bcl-xL and demonstrated how inherent structural disorder may provide a physical basis of protein regulated unfolding in signaling transduction. We have also constructed a series of efficient coarse-grained models to directly simulate the interactions between IDPs and unveiled how the preexisting structural elements accelerate binding of ACTR to NCBD by promoting efficient folding upon encounter. These studies shed important light on how IDPs perform functions in the cellular regulatory network, but also reveal the necessity of new sampling techniques for more efficient simulations of IDPs. We have thus developed a novel sampling technique, called multiscale enhanced sampling (MSES). MSES couples the atomistic model with coarse-grained ones, to accelerate the sampling of atomistic conformational space. Bias from coupling to a coarse-grained model can be removed using Hamiltonian replica exchange. To achieve the best possible efficiency of MSES simulations, we have developed a new hybrid resolution protein model that could capture the essential features of IDP structures, so as to generate local and long-range fluctuations that are largely consistent with those at the atomistic level. We have also developed an advanced replica exchange protocol, to allow the fast conformational transitions observed in the coupled conditions to be rapidly exchanged to the unbiased limit. Application of these strategies to characterize the structural ensembles of a few non-trivial IDPs shows that faster convergence rate can be achieved, demonstrating the great potential of MSES for atomistic simulations of larger and more complex IDPs

MCL-1 promiscuity and the structural resilience of its binding partners

The allosteric protein MCL-1 and its natural inhibitors, the BH3-only proteins PUMA, BIM, and NOXA regulate apoptosis by interacting promiscuously within an entangled binding network. Little is known about the transient processes and dynamic conformational fluctuations that are the basis for the formation and stability of the MCL-1/BH3-only complex. In this study, we designed photoswitchable versions of MCL-1/PUMA and MCL-1/NOXA, and investigated the protein response after an ultrafast photo-perturbation with transient infrared spectroscopy. We observed partial α-helical unfolding in all cases, albeit on strongly varying timescales (1.6 ns for PUMA, 9.7 ns for the previously studied BIM, and 85 ns for NOXA). These differences are interpreted as a BH3-only-specific “structural resilience” to defy the perturbation while remaining in MCL-1’s binding pocket. Thus, the presented insights could help to better understand the differences between PUMA, BIM, and NOXA, the promiscuity of MCL-1, in general, and the role of the proteins in the apoptotic network

From disorder to order: the importance of context in protein folding and binding mechanisms

Anfinsen’s seminal work has shown that the information required for a protein to fold into its specific three-dimensional structure is encoded into its amino acid sequence. The protein structure was believed to determine its activity, meaning that a protein needs to fold in order to function. More recently, intrinsically disordered proteins (IDPs) have been shown to represent a significant portion of the proteome. Despite the lack of a predefined structure, they still play important roles in cellular function, challenging the structure-function paradigm. Proteins are largely studied in isolated conditions, but in a cellular environment they are part of a vastly more complex system. The work presented here aims to shed light on how context can influence folding and binding mechanisms. First, we used SasG – a bacterial protein that defies the disorder prediction with its unique sequence composition and unusual structure – as a template to investigate co-translational folding, and how the presence of the ribosome can affect its folding mechanism. SasG in vitro translation was investigated utilising force profile experiments. We showed that both the G52 and E-G52 constructs can fold very early, when still inside the vestibule of the ribosome. Moreover, our results suggest that non-native interactions can also provide sufficient force to release the stall sequence. Next we employed protein members of the BCL-2 family – involved in controlling the cell death mechanism – to understand what encodes a coupled folding and binding reaction. Although displaying a variety of conformations, some IDPs can fold upon binding to a partner protein. Promiscuous binding is a great advantage of disordered molecules, as multiple IDPs are able to bind and fold to the same partner protein. This raises the question of what orchestrates a coupled folding and binding reaction: the IDP or the partner protein? Using phi-value analysis we studied four IDP-partner protein complexes, composed of alternative pairs of BCL-2 family members. In the bimolecular context, the disordered protein dictates the transition state interactions. Therefore, analogous to Anfinsen’s postulate, the folding pathway is encoded by the protein that folds, even when binding to another macromolecule is required. Finally, studies of the BCL-2 member BID on its full-length context showed that it cannot interact with its partner A1 unless it is cleaved (tBID). These results provide insights on the role of tBID as a player during programmed cell death and hence why the pathway of cleaving BID with caspase is energetically favourable