Flexible Intermediates During SH3 Binding

SH3 domains are the most common protein interaction domains and are found across all forms of life with at least 400 in humans alone. Theses domains often bind to flexible proteins known as intrinsically disordered proteins (IDPs). However, little is known about the binding mechanism between SH3 domains and their IDP binding partners which tend to be proline rich. One SH3 domain found in yeast, AbpSH3, has a binding site for the IDP ArkA. Molecular dynamics simulations were used to model the binding mechanism of AbpSH3 with ArkA. AbpSH3 is hypothesized to undergo a multi-step binding process with ArkA, beginning with the formation of an encounter complex where an ensemble of ArkA conformations are populated in an equilibrium exchange. The two halves of the ArkA sequence, segments 1 (N-terminal) and 2 (C-terminal), are also believed to bind independently. Segment 1, which contains the PxxP motif, is more structured than segment 2. We characterized the structural ensemble of ArkA alone. Then, we performed simulations of initial binding interactions between the SH3 domain and ArkA. The peptide was initially placed at least 10 Å away from the SH3 domain in explicit water. Upon binding, ArkA sampled a wider range of contacts with the domain, compared to simulations started from the bound structure. This suggests that ArkA is forming a flexible encounter complex with the SH3 domain as a binding intermediate. We also observe that the PxxP motif in segment 1 can bind to the AbpSH3 in both the forward and reverse orientation in the encounter ensemble. We saw agreement, within an order of magnitude, between the ArkA binding rate in our simulations and that determined from experimental data. In the future, we will explore the role of electrostatics in this binding interaction

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

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

分子動力学シミュレーションで探る天然変性タンパク質の準安定複合体構造と結合解離調節機構

天然変性タンパク質は従来の折りたたまれるタンパク質とは異なり，一定の立体構造に折りたたまれない。とてもやわらかい立体構造をもっと言い換えることができる。pKID(リン酸化 kinase-in-ducibledomain）は典型的な天然変性タンパク質である。pKID単体は生理的条件下で折りたたまれない。しかし， pKIDは結合相手タンパク質KIX(KID-interacting domain）との結合時には安定な一つの立体構造に折りたたまれる。この現象を結合と共役した折りたたみと呼ばれる。pKID-KIX複合体形成は実験的にもよく研究されており， pKID-KIX複合体構造は核磁気共鳴分光法によって特定されている。しかし，その最安定な複合体構造の他に準安定な複合体構造が示唆されていたが，その準安定な複合体構造の詳細は不明なままであった。そこで，詳細な計算モデルを用いて， pKIDのKIX結合状態における自由エネルギー地形の算出を実施した。自由エネルギ一地形とはある反応座標を軸にして，その軸上における自由エネルギ一変化を図示したものである。ここでは，計算構造と実験構造の差を反応座標にとり，pKID-KIX結合状態の自由エネルギー地形を求めた。その結果，準安定な領域を示し，その具体的な立体構造を示すことができた。さらに， pKIDのやわらかさがpKID-KIX間結合に与える影響を粗視化モデルを用いて明らかにした。pKID粗視化モデルに分子のやわらかさを導入し， 分子のやわらかさを変化させたときのpKID-KIX間結合解離速度および結合自由エネルギーの変化を求めた。その結果，分子のやわらかさは結合速度よりもむしろ解離速度を上昇させる効果が強く， pKID-KIX間結合自由エネルギーを上昇させることを示した。本稿では，ここで使用してきた計算モデルを簡単に紹介する。今後，天然変性タンパク質のような様々な立体構造を形成する“やわらかい”生体分子の立体構造集団と相互作用機構を解明する上で， 分子動力学シミュレーションは役立つと思われる。Article信州大学農学部紀要 53: 34-43(2017)departmental bulletin pape

ATOMISTIC SIMULATIONS OF INTRINSICALLY DISORDERED PROTEIN FOLDING AND DYNAMICS

Intrinsically disordered proteins (IDPs) are crucial in biology and human diseases, necessitating a comprehensive understanding of their structure, dynamics, and interactions. Atomistic simulations have emerged as a key tool for unraveling the molecular intricacies and establishing mechanistic insights into how these proteins facilitate diverse biological functions. However, achieving accurate simulations requires both an appropriate protein force field capable of describing the energy landscape of functionally relevant IDP conformations and sufficient conformational sampling to capture the free energy landscape of IDP dynamics. These factors are fundamental in comprehending potential IDP structures, dynamics, and interactions. I first conducted explicit solvent simulations to assess the performance of two state-of-the-art protein force fields, namely CHARMM36m and a99SB-disp, in capturing the stability of small protein-protein interactions. To evaluate their accuracy, I selected a set of 46 amino acid backbone and side chain pairs with representative configurations and computed the free energy profiles of their interactions. The results demonstrated that CHARMM36m consistently predicted stronger protein-protein interactions compared to a99SB-disp. Notably, the most significant overestimation in CHARMM36m occurred in charged pairs involving Arg and Glu side chains, with an overestimation of up to 2.9 kcal/mol. Through free energy decomposition analysis, I determined that these overestimations were primarily driven by protein-water electrostatic interactions rather than van der Waals (vdW) interactions. Consequently, these findings suggest that careful rebalancing of electrostatic interactions should be considered in the further optimization of protein force fields. In order to enhance the conformational sampling of IDPs, I developed an integrated approach that combines an improved implicit solvent model called Generalized Born with molecular volume and solvent accessible surface area (GBMV2/SA) with a multiscale enhanced sampling (MSES) technique. To make this approach more efficient, I implemented it as a standalone OpenMM plugin on Graphics Processing Units (GPUs). The results demonstrated that the GPU-GBMV2/SA model achieved numerical equivalence to the original CPU-GBMV2/SA models, while providing a remarkable ~60x speedup on a single NVIDIA TITAN X (Pascal) graphics card for molecular dynamic simulations of both folded and unstructured proteins. This significant acceleration greatly facilitated the application of the approach in biomolecular simulations. In addition, I conducted an evaluation of the reliability of GBMV2/SA models in simulating both folded and unfolded proteins. The results revealed that the GBMV2/SA model accurately describes small proteins, but its applicability is limited when it comes to larger proteins such as KID and p53-TAD proteins. This limitation can be attributed to the absence of long-range solute-solvent dispersion interactions in the model. To address this issue, I introduced a comprehensive treatment of nonpolar solvation free energy called GBMV2/NP model. Unfortunately, the GBMV2/NP model exhibited a destabilizing effect on well-folded proteins, particularly larger ones, due to an inaccurate representation of the repulsive solvent accessible surface area (SASA) model caused by the utilization of unphysical van der Waals volume. This observation highlights the need for further improvements in accurately describing the nonpolar term in the model

The significance of bioelectricity on all levels of organization of an organism. Part 1: From the subcellular level to cells

Bioelectricity plays an essential role in the structural and functional organization of biological organisms. In this first part of our multi-part series of articles, we summarise the importance of bioelectricity for the basic structural level of biological organization, i.e. from the subcellular level (charges, ion channels, molecules and cell organelles) to cells

Molecular Simulations of Pathways and Kinetics for Protein-protein Binding Processes

Protein-protein binding processes are crucial for biological functions and characterizing these processes fully has been a challenge in biophysics. In this work I use weighted ensemble path sampling method coupled with molecular simulations of varying levels of detail to answer long standing questions regarding protein-protein binding. In Chapter 3, I investigate the effects of preorganization on association between an intrinsically disordered peptide fragment of tumor suppressor p53 and the MDM2 protein using flexible residue level models. I simulated the binding process between p53 and MDM2 with varying degrees of preorganization in p53 and determined that the association rate constant of p53 peptide does not depend on the extent to which the peptide is preorganized for binding MDM2. In Chapter 4, I apply simulations with flexible molecular models to directly compute the “basal” kon for the association of the two proteins barnase and barstar, in the absence of electrostatics. I simulated the binding process between exact hydrophobic analogues barnase and barstar and determined the extent with which the electrostatics enhance the basal kon. Finally, in Chapter 5, I have generated binding pathways of barnase and barstar using all-atom simulations with explicit solvent. This study not only enabled a more detailed characterization of the binding mechanism but also provided an opportunity to determine the role of solvent in the binding process. Water molecules are proposed to play a crucial role in binding of barnase and barstar since water molecules can be found at the binding interface in the crystal structure and they increase the interfacial complementarity. Overall, the work presented here demonstrates the power of the weighted ensemble strategy in making it practical to characterize binding processes that are otherwise unfeasible for standard simulation

New experimental and theoretical tools for studying protein systems with elements of structural disorder

Disordered proteins are one class of proteins which do not possess well-folded three-dimensional structures as their native conformations. Many eukaryotic proteins have been found to be fully disordered or contain certain disordered regions. Disordered proteins usually display several characteristic properties, such as increased motional freedom and the conformational heterogeneity caused by that. The elements of structural disorder are commonly involved in many important biological functions and are implicated in many diseases. Therefore, the study of disordered proteins has become one of the most important research topics in recent years. This thesis presents results from three different research projects; the common feature is that all systems being studied contain varying amount of structural disorder. Most results have been obtained based on experimental nuclear magnetic resonance (NMR) studies and molecular dynamics (MD) simulations. Both are among the most popular biophysical techniques for studying molecular dynamics. The first project investigates the relationship between domain cooperativity and residual dipolar coupling (RDC) parameters based on a series of two-domain chimera proteins with disordered linkers. Many eukaryotic proteins contain multiple domains and their biological functions are closely related to the property of domain cooperativity, which is often regulated by the linker region. Therefore it is necessary to develop suitable tools to characterize linker region properties in order to better understand biological functions of multidomain proteins. The second project is about the development of NMR pulse sequences for studying disordered proteins. Two new NMR pulse sequences, PD-CPMG and CP-HISQC, have been developed. Both experiments are well suited for studying intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) under physiological conditions. These two experiments produce higher precision for 15N R2 rates measurement or higher sensitivity in 1H– 15N HSQC spectra respectively. Besides, they also show many advantages over most other existing experiments for studying IDPs. The last project is about protein-peptide encounter complex study based on Crk-Sos model system. The ten-residue Sos peptide serves as a minimal model for disordered proteins. Encounter complex is an important type of intermediate state formed during many protein interactions. Such complexes are usually characterized by a large amount of motional freedom and conformational heterogeneity. Therefore their properties are considerably different from tight-binding complexes which are more commonly studied. Although it is usually quite difficult to study encounter complexes using standard biophysical techniques, in this project we have successfully characterized structural and dynamic properties of Crk-Sos electrostatic encounter complex with a combination of MD simulations and experimental NMR approaches. It can be directly seen from the structural model based on MD trajectories that Sos peptide in the encounter complex remains highly dynamic, sampling large area on the surface of Crk N-SH3 domain. Such strategy can also be utilized for studying many other encounter complexes involving disordered proteins or peptide

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