Conformational Substates Research Articles

Assessing AF2’s ability to predict structural ensembles of proteins

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined. Additionally, molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction and compare the obtained structures with ensembles obtained from MD simulations and NMR. We provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. Significant minima of the free energy surfaces remain undetected.

Magnesium induced structural reorganization in the active site of adenylate kinase.

Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Experimental Evidence for the Role of Dynamics in pH-Dependent Enzymatic Activity.

Enzymatic activity is heavily influenced by pH, but the rationale for the dynamical mechanism of pH-dependent enzymatic activity has not been fully understood. In this work, combined neutron scattering techniques, including quasielastic neutron scattering (QENS) and small angle neutron scattering (SANS), are used to study the structural and dynamic changes of a model enzyme, xylanase, under different pH and temperature environments. The QENS results reveal that xylanase at optimal pH exhibits faster relaxational dynamics and a lower energy barrier between conformational substates. The SANS results demonstrate that pH affects both xylanase's stability and monodispersity. Our findings indicate that enzymes have optimized stability and function under their optimal pH conditions, with both structure and dynamics being affected. The current study offers valuable insights into enzymatic functionality mechanisms, allowing for broad industrial applications.

Structural evolution of the conformational ensembles in peptide fibrillar aggregates

Exploring the folding structures and intermolecular recognitions of proteins in their assemblies is essential for revealing the mechanisms of amyloid fibrillar aggregation. However, the molecular-level description of protein structural changes with aggregation progression is challenging due to the heterogeneity of assembled structures. Here we report the use of scanning tunneling microscopy (STM), which is competent in analyzing the highly heterogeneous structures with submolecular resolution, to probe the structures of human islet amyloid polypeptide (hIAPP) assemblies at the different time points of hIAPP fibrillation. The conformational substate ensembles of the β-strands formed by hIAPP as well as the interpeptide interactions are found to change with time in the growth and plateau phases. Detailed analyses of the distribution probability of interpeptide interactions at the different time points manifest that the enthalpic contribution to the structural conversions of β-sheet fibrillation becomes more significant. Our single-molecular level studies highlight the dynamic nature of protein structures within a β-sheet-assembled fibril.

The Role of Force Fields and Water Models in Protein Folding and Unfolding Dynamics.

Protein folding is a fascinating, not fully understood phenomenon in biology. Molecular dynamics (MD) simulations are an invaluable tool to study conformational changes in atomistic detail, including folding and unfolding processes of proteins. However, the accuracy of the conformational ensembles derived from MD simulations inevitably relies on the quality of the underlying force field in combination with the respective water model. Here, we investigate protein folding, unfolding, and misfolding of fast-folding proteins by examining different force fields with their recommended water models, i.e., ff14SB with the TIP3P model and ff19SB with the OPC model. To this end, we generated long conventional MD simulations highlighting the perks and pitfalls of these setups. Using Markov state models, we defined kinetically independent conformational substates and emphasized their distinct characteristics, as well as their corresponding state probabilities. Surprisingly, we found substantial differences in thermodynamics and kinetics of protein folding, depending on the combination of the protein force field and water model, originating primarily from the different water models. These results emphasize the importance of carefully choosing the force field and the respective water model as they determine the accuracy of the observed dynamics of folding events. Thus, the findings support the hypothesis that the water model is at least equally important as the force field and hence needs to be considered in future studies investigating protein dynamics and folding in all areas of biophysics.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).

Alternate conformations found in protein structures implies biological functions: A case study using cyclophilin A

Protein dynamics linked to numerous biomolecular functions, such as ligand binding, allosteric regulation, and catalysis, must be better understood at the atomic level. Reactive atoms of key residues drive a repertoire of biomolecular functions by flipping between alternate conformations or conformational substates, seldom found in protein structures. Probing such sparsely sampled alternate conformations would provide mechanistic insight into many biological functions. We are therefore interested in evaluating the instance of amino acids adopted alternate conformations, either in backbone or side-chain atoms or in both. Accordingly, over 70000 protein structures appear to contain alternate conformations only 'A' and 'B' for any atom, particularly the instance of amino acids that adopted alternate conformations are more for Arg, Cys, Met, and Ser than others. The resulting protein structure analysis depicts that amino acids with alternate conformations are mainly found in the helical and β-regions and are often seen in high-resolution X-ray crystal structures. Furthermore, a case study on human cyclophilin A (CypA) was performed to explain the pre-existing intrinsic dynamics of catalytically critical residues from the CypA and how such intrinsic dynamics perturbed upon Ser99Thr mutation using molecular dynamics simulations on the ns-μs timescale. Simulation results demonstrated that the Ser99Thr mutation had impaired the alternate conformations or the catalytically productive micro-environment of Phe113, mimicking the experimentally observed perturbation captured by X-ray crystallography. In brief, a deeper comprehension of alternate conformations adopted by the amino acids may shed light on the interplay between protein structure, dynamics, and function.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

Simulated pressure changes in LacI suggest a link between hydration and functional conformational changes

The functions of many proteins are associated with interconversions among conformational substates. However, these substates can be difficult to measure experimentally, and determining contributions from hydration changes can be especially difficult. Here, we assessed the use of pressure perturbations to sample the substates accessible to the Escherichia coli lactose repressor protein (LacI) in various liganded forms. In the presence of DNA, the regulatory domain of LacI adopts an Open conformation that, in the absence of DNA, changes to a Closed conformation. Increasing the simulation pressure prevented the transition from an Open to a Closed conformation, in a similar manner to the binding of DNA and anti-inducer, ONPF. The results suggest the hydration of specific residues play a significant role in determining the population of different LacI substates and that simulating pressure perturbation could be useful for assessing the role of hydration changes that accompany functionally-relevant amino acid substitutions.

Kinetic Resolution of the Atomic 3D Structures Formed by Ground and Excited Conformational States in an RNA Dynamic Ensemble.

Knowing the 3D structures formed by the various conformations populating the RNA free-energy landscape, their relative abundance, and kinetic interconversion rates is required to obtain a quantitative and predictive understanding of how RNAs fold and function at the atomic level. While methods integrating ensemble-averaged experimental data with computational modeling are helping define the most abundant conformations in RNA ensembles, elucidating their kinetic rates of interconversion and determining the 3D structures of sparsely populated short-lived RNA excited conformational states (ESs) remains challenging. Here, we developed an approach integrating Rosetta-FARFAR RNA structure prediction with NMR residual dipolar couplings and relaxation dispersion that simultaneously determines the 3D structures formed by the ground-state (GS) and ES subensembles, their relative abundance, and kinetic rates of interconversion. The approach is demonstrated on HIV-1 TAR, whose six-nucleotide apical loop was previously shown to form a sparsely populated (∼13%) short-lived (lifetime ∼ 45 μs) ES. In the GS, the apical loop forms a broad distribution of open conformations interconverting on the pico-to-nanosecond time scale. Most residues are unpaired and preorganized to bind the Tat-superelongation protein complex. The apical loop zips up in the ES, forming a narrow distribution of closed conformations, which sequester critical residues required for protein recognition. Our work introduces an approach for determining the 3D ensemble models formed by sparsely populated RNA conformational states, provides a rare atomic view of an RNA ES, and kinetically resolves the atomic 3D structures of RNA conformational substates, interchanging on time scales spanning 6 orders of magnitude, from picoseconds to microseconds.

Signature of functional enzyme dynamics in quasielastic neutron scattering spectra: The case of phosphoglycerate kinase.

We present an analysis of high-resolution quasi-elastic neutron scattering spectra of phosphoglycerate kinase which elucidates the influence of the enzymatic activity on the dynamics of the protein. We show that in the active state the inter-domain motions are amplified and the intra-domain asymptotic power-law relaxation ∝t-α is accelerated, with a reduced coefficient α. Employing an energy landscape picture of protein dynamics, this observation can be translated into a widening of the distribution of energy barriers separating conformational substates of the protein.

Specifying conformational heterogeneity of multi-domain proteins at atomic resolution

The conformational landscape of multi-domain proteins is inherently linked to their specific functions. This also holds for polyubiquitin chains that are assembled by two or more ubiquitin domains connected by a flexible linker thus showing a large interdomain mobility. However, molecular recognition and signal transduction are associated with particular conformational substates that are populated in solution. Here, we apply high-resolution NMR spectroscopy in combination with dual-scale MD simulations to explore the conformational space of K6-, K29-, and K33-linked diubiquitin molecules. The conformational ensembles are evaluated utilizing a paramagnetic cosolute reporting on solvent exposure plus a set of complementary NMR parameters. This approach unravels a conformational heterogeneity of diubiquitins and explains the diversity of structural models that have been determined for K6-, K29-, and K33-linked diubiquitins in free and ligand-bound states so far. We propose a general application of the approach developed here to demystify multi-domain proteins occurring in nature.

Combining molecular dynamics simulations and scoring method to computationally model ubiquitylated linker histones in chromatosomes.

The chromatin in eukaryotic cells plays a fundamental role in all processes during a cell's life cycle. This nucleoprotein is normally tightly packed but needs to be unpacked for expression and division. The linker histones are critical for such packaging processes and while most experimental and simulation works recognize their crucial importance, the focus is nearly always set on the nucleosome as the basic chromatin building block. Linker histones can undergo several modifications, but only few studies on their ubiquitylation have been conducted. Mono-ubiquitylated linker histones (HUb), while poorly understood, are expected to influence DNA compaction. The size of ubiquitin and the globular domain of the linker histone are comparable and one would expect an increased disorder upon ubiquitylation of the linker histone. However, the formation of higher order chromatin is not hindered and ubiquitylation of the linker histone may even promote gene expression. Structural data on chromatosomes is rare and HUb has never been modeled in a chromatosome so far. Descriptions of the chromatin complex with HUb would greatly benefit from computational structural data. In this study we generate molecular dynamics simulation data for six differently linked HUb variants with the help of a sampling scheme tailored to drive the exploration of phase space. We identify conformational sub-states of the six HUb variants using the sketch-map algorithm for dimensionality reduction and iterative HDBSCAN for clustering on the excessively sampled, shallow free energy landscapes. We present a highly efficient geometric scoring method to identify sub-states of HUb that fit into the nucleosome. We predict HUb conformations inside a nucleosome using on-dyad and off-dyad chromatosome structures as reference and show that unbiased simulations of HUb produce significantly more fitting than non-fitting HUb conformations. A tetranucleosome array is used to show that ubiquitylation can even occur in chromatin without too much steric clashes.

Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite β-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.

Single-molecule visualization determines conformational substate ensembles in β-sheet–rich peptide fibrils

An understanding of protein conformational ensembles is essential for revealing the underlying mechanisms of interpeptide recognition and association. However, experimentally resolving multiple simultaneously existing conformational substates remains challenging. Here, we report the use of scanning tunneling microscopy (STM) to analyze the conformational substate ensembles of β sheet peptides with a submolecular resolution (in-plane <2.6 Å). We observed ensembles of more than 10 conformational substates (with free energy fluctuations between several k B T s) in peptide homoassemblies of keratin (KRT) and amyloidal peptides (−5Aβ42 and TDP-43 341–357). Furthermore, STM reveals a change in the conformational ensemble of peptide mutants, which is correlated with the macroscopic properties of peptide assemblies. Our results demonstrate that the STM-based single-molecule imaging can capture a thorough picture of the conformational substates with which to build an energetic landscape of interconformational interactions and can rapidly screen conformational ensembles, which can complement conventional characterization techniques.

Experimental Insights into Conformational Ensembles of Assembled β-Sheet Peptides.

Deciphering the conformations and interactions of peptides in their assemblies offers a basis for guiding the rational design of peptide-assembled materials. Here we report the use of scanning tunneling microscopy (STM), a single-molecule imaging method with a submolecular resolution, to distinguish 18 types of coexisting conformational substates of the β-strand of the 8-37 segment of human islet amyloid polypeptide (hIAPP 8-37). We analyzed the pairwise peptide-peptide interactions in the hIAPP 8-37 assembly and found 82 interconformation interactions within a free energy difference of 3.40 kBT. Besides hIAPP 8-37, this STM method validates the existence of multiple conformations of other β-sheet peptide assemblies, including mutated hIAPP 8-37 and amyloid-β 42. Overall, the results reported in this work provide single-molecule experimental insights into the conformational ensemble and interpeptide interactions in the β-sheet peptide assembly.

Parameterization of the miniPEG-Modified γPNA Backbone: Toward Induced γPNA Duplex Dissociation.

γ-Modified peptide nucleic acids (γPNAs) serve as potential therapeutic agents against genetic diseases. Miniature poly(ethylene glycol) (miniPEG) has been reported to increase solubility and binding affinity toward genetic targets, yet details of γPNA structure and dynamics are not understood. Within our work, we parameterized missing torsional and electrostatic terms for the miniPEG substituent on the γ-carbon atom of the γPNA backbone in the CHARMM force field. Microsecond timescale molecular dynamics simulations were carried out on six miniPEG-modified γPNA duplexes from NMR structures (PDB ID: 2KVJ). Three NMR models for the γPNA duplex (PDB ID: 2KVJ) were simulated as a reference for structural and dynamic changes captured for the miniPEG-modified γPNA duplex. Principal component analysis performed on the γPNA backbone atoms identified a single isotropic conformational substate (CS) for the NMR simulations, whereas four anisotropic CSs were identified for the ensemble of miniPEG-modified γPNA simulations. The NMR structures were found to have a 23° helical bend toward the major groove, consistent with our simulated CS structure of 19.0°. However, a significant difference between simulated methyl- and miniPEG-modified γPNAs involved the opportunistic invasion of miniPEG through the minor and major groves. Specifically, hydrogen bond fractional analysis showed that the invasion was particularly prone to affect the second G-C base pair, reducing the Watson-Crick base pair hydrogen bond by 60% over the six simulations, whereas the A-T base pairs decreased by only 20%. Ultimately, the invasion led to base stack reshuffling, where the well-ordered base stacking was reduced to segmented nucleobase stacking interactions. Our 6 μs timescale simulations indicate that duplex dissociation suggests the onset toward γPNA single strands, consistent with the experimental observation of decreased aggregation. To complement the insight of miniPEG-modified γPNA structure and dynamics, the new miniPEG force field parameters allow for further exploration of such modified γPNA single strands as potential therapeutic agents against genetic diseases.

Visualization and characterization of the elusive MHC I intermediate states by x-ray crystallography and in silicostudies

Abstract Background: Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. It is the molecular features of these intermediates that are recognized by tapasin and TAPBPR at the C-terminus of the groove. To date, however, MHC I intermediates remain elusive molecules due to their transient nature. Moreover, whether the N-terminus of the groove has a role in MHC I maturation, and if it also serves as a conformationally sensing region, is not well characterized. Methods: We designed the HLA-B8-restricted 20mer (RA) 6FAKKKYCL and (RA) 6FVKKKYCL peptides and corresponding 8mer FAKKKYCL and FVKKKYCL controls. We determined the x-ray crystal structures of HLA-B8E76C presenting these four peptides. Molecular dynamics (MD) simulations of the complexes were also performed. Results: The structures of FA and FV 20mers revealed significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal MHC I residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also showed highly unusual features in MHC I and peptides at the N-terminus of the groove, and in MHC I-peptide interaction. MD simulations showed that these complexes have varying degrees of mobility in a manner consistent with our structures. We suggest that our 20mer structures represent conformational substates explored by MHC I during peptide selection. Conclusion: Our study advances our understanding of peptide-induced conformational adaptation and functional dynamics in MHC I, and brings into focus the N-terminus of the groove as an important region in mechanisms of high-affinity peptide selection. NIAID, National Institutes of Health (R01 AI114467, R01 AI108546, and R21 AI173863)

Comparative Study of High-Resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions

The treatment of insulin-dependent diabetes mellitus is characterized by artificial supplementation of pancreatic β-cell ability to regulate sugar levels in the blood. Even though various insulin analogs are crucial for reasonable glycemic control, understanding the dynamic mechanism of the insulin analogs may help to improve the best-protracted insulin analog to assist people with type 1 diabetes (T1D) to live comfortably while maintaining tight glycemic control. Here, we present the high-resolution crystal structure of NN304, known as insulin detemir, to 1.7 Å resolution at cryogenic temperature. We computationally further investigated our crystal structure’s monomeric-dimeric conformation and dynamic profile by comparing it with a previously available detemir structure (PDB ID: 1XDA). Our structure (PDB ID: 8HGZ), obtained at elevated pH, provides electrostatically triggered minor movements in the equilibrium between alternate conformational substates compared to the previous structure, suggesting it might induce an intermediate state in the dissociation pathway of the insulin detemir’s hexamer:dihexamer equilibrium. Supplemented with orientational cross-correlation analysis by a Gaussian network model (GNM), this alternate oligomeric conformation offers the distinct cooperative motions originated by loose coupling of distant conformational substates of a protracted insulin analog that has not been previously observed.