Recognition of protein allosteric states and residues: Machine learning approaches

Allostery is a process by which proteins transmit the effect of perturbation at one site to a distal functional site upon certain perturbation. As an intrinsically global effect of protein dynamics, it is difficult to associate protein allostery with individual residues, hindering effective selection of key residues for mutagenesis studies. The machine learning models including decision tree (DT) and artificial neural network (ANN) models were applied to develop classification model for a cell signaling allosteric protein with two states showing extremely similar tertiary structures in both crystallographic structures and molecular dynamics simulations. Both DT and ANN models were developed with 75% and 80% of predicting accuracy, respectively. Good agreement between machine learning models and previous experimental as well as computational studies of the same protein validates this approach as an alternative way to analyze protein dynamics simulations and allostery. In addition, the difference of distributions of key features in two allosteric states also underlies the population shift hypothesis of dynamics‐driven allostery model. © 2018 Wiley Periodicals, Inc.     

Resonance energy transfer and ligand binding studies on pH-induced folded states of human serum albumin

Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster’s resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.     

Allostery in a disordered protein: oxidative modifications to α-synuclein act distally to regulate membrane binding

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson's disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ～95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with the lipid bilayer. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane-binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function.     

CEESY: characterizing the conformation of unobservable protein states

Protein conformations that are only marginally populated often play important roles as intermediate states in many processes such as ligand binding, enzyme catalysis, allostery, and protein folding. An NMR method is presented that can give valuable information about the structure of these "excited states" by measuring the relative position of exchanging excited- and ground-state resonances using a single 2D spectrum. This new approach can be applied to any nucleus, which will facilitate a complete structural characterization of these states.     

Using steered molecular dynamics to study the interaction between ADP and the nucleotide-binding domain of yeast Hsp70 protein Ssa1

Genetics experiments have identified six mutations located in the subdomain IA (A17V, R23H, G32D, G32S, R34K, V372I) of Ssa1 that influence propagation of the yeast [PSI⁺] prion. However, the underlining molecular mechanisms of these mutations are still unclear. The six mutation sites are present in the IA subdomain of the nucleotide-binding domain (NBD). The ATPase subdomain IA is a critical mediator of inter-domain allostery in Hsp70 molecular chaperones, so the mutation and changes in this subdomain may influence the function of the substrate-binding domain. In addition, ADP release is a rate-limiting step of the ATPase cycle and dysregulation of the ATPase cycle influences the propagation of the yeast [PSI⁺] prion. In this work, steered molecular dynamics (SMD) simulations were performed to explore the interaction between ADP and NBD. Results suggest that during the SMD simulations, hydrophobic interactions are predominant and variations in the binding state of ADP within the mutants is a potential reason for in vivo effects on yeast [PSI⁺] prion propagation. Additionally, we identify the primary residues in the ATPase domain that directly constitute the main hydrophobic interaction network and directly influence the ADP interaction state with the NBD of Ssa1. Furthermore, this in silico analysis reaffirms the importance of previously experimentally-determined residues in the Hsp70 ATPase domain involved in ADP binding and also identifies new residues potentially involved in this process.     

Alchemical Grid Dock (AlGDock): Binding Free Energy Calculations between Flexible Ligands and Rigid Receptors

Alchemical Grid Dock (AlGDock) is open-source software designed to compute the binding potential of mean force—the binding free energy between a flexible ligand and a rigid receptor—for a small organic ligand and a biological macromolecule. Multiple BPMFs can be used to rigorously compute binding affinities between flexible partners. AlGDock uses replica exchange between thermodynamic states at different temperatures and receptor–ligand interaction strengths. Receptor–ligand interaction energies are represented by interpolating precomputed grids. Thermodynamic states are adaptively initialized and adjusted on-the-fly to maintain adequate replica exchange rates. In demonstrative calculations, when the bound ligand is treated as fully solvated, AlGDock estimates BPMFs with a precision within 4 kT in 65% and within 8 kT for 91% of systems. It correctly identifies the native binding pose in 83% of simulations. Performance is sometimes limited by subtle differences in the important configuration space of sampled and targeted thermodynamic states. © 2019 Wiley Periodicals, Inc.     

Structural-dynamical investigation of the ZnuA histidine-rich loop: involvement in zinc management and transport

Comparative homology modelling techniques have been used to model the protein ZnuA from Salmonella enterica serovar Typhimurium using the 3D structure of the homologous protein from Escherichia coli. These two-domain proteins bind one Zn²⁺ atom, with high affinity, in the inter-domain cleft and possess a histidine-rich loop in the N-terminal domain. Alternative structures of the ZnuA histidine-rich loop, never resolved by the X-ray diffraction method, have been modelled. A model of the apo form, one with the histidine-rich loop deleted and two alternative structures with a second zinc ion bound to the histidine-rich loop, have been generated. In all the modelled proteins, investigated through molecular dynamics simulation, the histidine-rich loop is highly mobile and its fluctuations are correlated to the ligand stability observed in the zinc sites. Based on the plasticity of the histidine-rich loop and its significant effects on protein mobility a possible role in the capture and/or transfer of the zinc ions has been suggested.     

分子动力学模拟研究点突变(Met108→Leu108)对树胶醛糖结合蛋白与配体作用的影响

为了研究点突变(Met108→Leu108)对树胶醛糖结合蛋白(ABP)与配体结合能力的影响,对ABP、ABP结合树胶醛糖复合物及ABP结合半乳糖复合物以及它们各自的突变体分别进行60 ns的分子动力学模拟.模拟结果表明,108号残基突变前后,电子等排体的两个氨基酸残基,使蛋白与配体间的范德华相互作用发生明显变化,同时导致蛋白的内部运动也发生变化,进而影响蛋白与配体的相互作用.进一步分析表明,突变前后的蛋白构象变化都趋向于两个结构域张开,而与配体的结合可减缓张开程度.     

Mapping allostery through equilibrium perturbation NMR spectroscopy

The understanding of allostery relies on the comparative analysis of macromolecules in their free and bound states. However, the direct free versus bound comparison is often challenging due to the instability of one of the two forms. This problem is effectively circumvented by using minor free/bound equilibrium perturbations which are tolerated without compromising sample stability. The subtle equilibrium perturbations are still able to reveal significant apo/holo differences if monitored by NMR experiments that are sensitive to minor populations within dynamic equilibria, such as NMR relaxation dispersion (NMRD) and hydrogen exchange (H/D and H/H) rates. These measurements are complementary to each other as they unmask how a ligand affects both the stable and the excited states of the free energy landscape for its protein receptor. The proposed equilibrium perturbation approach therefore significantly expands the scope of applicability of NMRD and hydrogen exchange experiments to the investigation of ligand-protein interactions, in general, unveiling allosteric "hot spot" maps for systems that have been traditionally elusive to direct free/bound comparisons.     

Ubiquitylation of Fe65 adaptor protein by neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) via the WW domain interaction with Fe65

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (⁷²PPLP⁷⁵) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (⁷²PPLP⁷⁵ was changed to ⁷²APLA⁷⁵) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.     

On the Role of Flexibility in Protein–Ligand Interactions: the Example of p53 Tetramerization Domain

The recognition of protein surfaces by designed ligands has become an attractive approach in drug discovery. However, the variable nature and irregular behavior of protein surfaces defy this new area of research. The easy to understand “lock‐and‐key” model is far from being the ideal paradigm in biomolecular interactions and, hence, any new finding on how proteins and ligands behave in recognition events paves a step of the way. Herein, we illustrate a clear example on how an increase in flexibility of both protein and ligand can result in an increase in the stability of the macromolecular complex. The biophysical study of the interaction between a designed flexible tetraguanidinium‐calix[4]arene and the tetramerization domain of protein p53 (p53TD) and its natural mutant p53TD‐R337H shows how the floppy mutant domain interacts more tightly with the ligand than the well‐packed wild‐type protein. Moreover, the flexible calixarene ligand interacts with higher affinity to both wild‐type and mutated protein domains than a conformationally rigid calixarene analog previously reported. These findings underscore the crucial role of flexibility in molecular recognition processes, for both small ligands and large biomolecular surfaces.     

Inter-domain linker prediction using amino acid compositional index

Protein chains are generally long and consist of multiple domains. Domains are distinct structural units of a protein that can evolve and function independently. The accurate and reliable prediction of protein domain linkers and boundaries is often considered to be the initial step of protein tertiary structure and function predictions. In this paper, we introduce CISA as a method for predicting inter-domain linker regions solely from the amino acid sequence information. The method first computes the amino acid compositional index from the protein sequence dataset of domain-linker segments and the amino acid composition. A preference profile is then generated by calculating the average compositional index values along the amino acid sequence using a sliding window. Finally, the protein sequence is segmented into intervals and a simulated annealing algorithm is employed to enhance the prediction by finding the optimal threshold value for each segment that separates domains from inter-domain linkers. The method was tested on two standard protein datasets and showed considerable improvement over the state-of-the-art domain linker prediction methods.     

Allosterically Activated Protein Self‐Assembly for the Construction of Helical Microfilaments with Tunable Helicity

Protein allostery, a chemical‐to‐mechanical effect that can precisely regulate protein structure, exists in many proteins. Herein, we demonstrate that protein allostery can be used to drive self‐assembly for the construction of tunable protein architectures. Calmodulin (CaM) was chosen as a model allosteric protein. Ca²⁺‐mediated contraction of CaM to a closed state can activate CaM and its ligand to self‐assemble into a 1D protein helical microfilament. Conversely, relaxation of CaM to the open state can unwind and further dissociate the helical assemblies. Fine regulation of the protein conformation by tuning the external Ca²⁺ level allows us to obtain various protein helical nanostructures with tunable helicity. This study offers a new approach toward chemomechanically controlled protein self‐assembly.     

Effect of Site-Specific O-Glycosylation on the Structural Behavior of NOTCH1 Receptor Extracellular EGF-like Domains 11 and 10

Human NOTCH1 receptor contains 36 epidermal growth factor (EGF)-like repeating domains, in which O-glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O-glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor–ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O-glycosylation patterns on the pivotal EGF-like repeats 10, 11, and 12, we performed chemical synthesis and NMR studies of site-specifically O-glycosylated EGF11 and EGF10. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O-glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type “sugar bridges” crosslinking Thr-O-GlcNAc in the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 and an amino acid in the hinge region between the domains, 445Thr-O-GlcNAc—IIe451 in domain 11 and 405Thr-O-GlcNAc—Gln411 in domain 10, respectively.     

Modulating Mobility: a Paradigm for Protein Engineering?

Proteins are highly mobile structures. In addition to gross conformational changes occurring on, for example, ligand binding, they are also subject to constant thermal motion. The mobility of a protein varies through its structure and can be modulated by ligand binding and other events. It is becoming increasingly clear that this mobility plays an important role in key functions of proteins including catalysis, allostery, cooperativity, and regulation. Thus, in addition to an optimum structure, proteins most likely also require an optimal dynamic state. Alteration of this dynamic state through protein engineering will affect protein function. A dramatic example of this is seen in some inherited metabolic diseases where alternation of residues distant from the active site affects the mobility of the protein and impairs function. We postulate that using molecular dynamics simulations, experimental data or a combination of the two, it should be possible to engineer the mobility of active sites. This may be useful in, for example, increasing the promiscuity of enzymes. Thus, a paradigm for protein engineering is suggested in which the mobility of the active site is rationally modified. This might be combined with more “traditional” approaches such as altering functional groups in the active site.     

新型室温熔盐二(三氟甲基磺酸酰)亚胺锂-乙酰胺体系的谱学研究

从分析二(三氟甲基磺酸酰)亚胺锂(LiTFSI)与乙酰胺形成熔盐的作用机制出发,通过红外和拉曼光谱的谱学分析并应用非局部密度泛函方法进行量化计算来对二者的相互作用进行了讨论.发现乙酰胺通过Li—O键与LiTFSI中Li+配位而破坏了LiTFSI的离子键,形成很大的配位阳离子,且正电荷被屏蔽在乙酰胺分子中;而TFSI-离子中电荷的部分离域导致电荷被终端—CF3基团屏蔽在整个分子中,这样两个大的阴阳离子间的库伦作用很弱;同时Li—O配位也导致乙酰胺分子间的氢键断裂,因而室温下体系以液体状态稳定存在.