Deciphering S100B Allosteric Signaling: The Role of a Peptide Target, TRTK-12, as an Ensemble Modulator.

Allostery is an essential biological phenomenon in which perturbation at one site in a biomolecule elicits a functional response at a distal location(s). It is integral to biological processes, such as cellular signaling, metabolism, and transcription regulation. Understanding allostery is also crucial for rational drug discovery. In this work, we focus on an allosteric S100B protein that belongs to the S100 class of EF-hand Ca2+-binding proteins. The Ca2+-binding affinity of S100B is modulated allosterically by TRTK-12 peptide binding 25 Å away from the Ca2+-binding site. We investigated S100B allostery by carrying out nuclear magnetic resonance (NMR) measurements along with microsecond-long molecular dynamics (MD) simulations on S100B/Ca2+ with/without TRTK-12 at different NaCl salt concentrations. NMR HSQC results show that TRTK-12 reorganizes how S100B/Ca2+ responds to different salt concentrations at both orthosteric and allosteric sites. The MD data suggest that TRTK-12 breaks the dynamic aromatic and hydrogen-bond interactions (not observed in X-ray crystallographic structures) between the hinge/helix and Ca2+-binding EF-hand loop of the two subunits in the homodimeric protein. This triggers rearrangement in the protein network architectures and leads to allosteric communication. Finally, computational studies of S100B at distinct ionic strengths suggest that ligand-bound species are more robust to the changing environment relative to the S100B/Ca2+ complex.

Modulation of Aß 16-22 aggregation by glucose.

The self-assembly of amyloid-beta (Aß) peptides into fibrillar structures in the brain is a signature of Alzheimer's disease. Recent studies have reported correlations between Alzheimer's disease and type-2 diabetes. Structurally, hyperglycemia induces covalent protein crosslinkings by advanced glycation end products (AGE), which can affect the stability of Aß oligomers. In this work, we leverage physics-based coarse-grained molecular simulations to probe alternate thermodynamic pathways that affect peptide aggregation propensities at varying concentrations of glucose molecules. Similar to previous experimental reports, our simulations show a glucose concentration-dependent increase in Aß aggregation rates, without changes in the overall secondary structure content. We discovered that glucose molecules prefer partitioning onto the aggregate-water interface at a specific orientation, resulting in a loss of molecular rotational entropy. This effectively hastens the aggregation rates, as peptide self-assembly can reduce the available surface area for peptide-glucose interactions. This work introduces a new thermodynamic-driven pathway, beyond chemical cross-linking, that can modulate Aß aggregation.

Unravelling heparin's enhancement of amyloid aggregation in a model peptide system.

A coarse-grained (CG) model for heparin, an anionic polysaccharide, was developed to investigate the mechanisms of heparin's enhancement of fibrillation in many amyloidogenic peptides. CG molecular dynamics simulations revealed that heparin, by forming contacts with the model amyloidogenic peptide, amyloid-ß's K16LVFFAE22 fragment (Aß16-22), promoted long-lived and highly beta-sheet-like domains in the peptide oligomers. Concomitantly, heparin-Aß16-22 contacts suppressed the entropy of mixing of the oligomers' beta-domains. Such oligomers could make better seeds for fibrillation, potentially contributing to heparin's fibril-enhancing behaviour. Additionally, reductions in heparin's flexibility led to delayed aggregation, and less ordered Aß16-22 oligomers, thus offering insights into the contrasting inhibition of fibrillation by the relatively rigid polysaccharide, chitosan.

Opposing roles of organic salts on mini-protein structure.

We investigated the effects of 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]) and choline chloride ([Chol][Cl]) on the local environment and conformational landscapes of Trp-cage and Trpzip4 mini-proteins using experimental and computational approaches. Fluorescence experiments and computational simulations revealed distinct behaviors of the mini-proteins in the presence of these organic salts. [EMIM][Cl] showed a strong interaction with Trp-cage, leading to fluorescence quenching and destabilization of its native structural interactions. Conversely, [Chol][Cl] had a negligible impact on Trp-cage fluorescence at low concentrations but increased it at high concentrations, indicating a stabilizing role. Computational simulations elucidated that [EMIM][Cl] disrupted the hydrophobic core packing and decreased proline-aromatic residue contacts in Trp-cage, resulting in a more exposed environment for Trp residues. In contrast, [Chol][Cl] subtly influenced the hydrophobic core packing, creating a hydrophobic environment near the tryptophan residues. Circular dichroism experiments revealed that [Chol][Cl] stabilized the secondary structure of both mini-proteins, although computational simulations did not show significant changes in secondary content at the explored concentrations. The simulations also demonstrated a more rugged free energy landscape for Trp-cage and Trpzip4 in [EMIM][Cl], suggesting destabilization of the tertiary structure for Trp-cage and secondary structure for Trpzip4. Similar fluorescence trends were observed for Trpzip4, with [EMIM][Cl] quenching fluorescence and exhibiting stronger interaction, while [Chol][Cl] increased the fluorescence at high concentrations. These findings highlight the interplay between [EMIM][Cl] and [Chol][Cl] with the mini-proteins and provide a detailed molecular-level understanding of how these organic salts impact the nearby surroundings and structural variations. Understanding such interactions is valuable for diverse applications, from biochemistry to materials science.

Mechanistic insights into the inhibition of amyloid-ß aggregation by chitosan.

Neurodegeneration related to Alzheimer's disease has long been linked to the accumulation of abnormal aggregates of amyloid-ß (Aß) peptides. Pre-fibrillar oligomeric intermediates of Aß aggregation are considered the primary drivers of neurotoxicity, however, their targetting remains an unresolved challenge. In response, the effects of macromolecular components of the blood-brain barrier, artificial extracellular matrix mimics, and polymeric drug delivery particles, on the aggregation of Aß peptides are gaining interest. Multiple experimental studies have demonstrated the potential of one such macromolecule, chitosan (CHT) - a polysaccharide with acid induced cationicity (pKa 6.5) - to inhibit the aggregation of Aß, and reduce the associated neurotoxic effects. However, the mechanistic details of this inhibitory action, and the structural details of the emergent Aß complexes are not understood. In this work, we probed how CHT modulated the aggregation of Aß's central hydrophobic core fragment, K16LVFFAE22, using coarse-grained molecular dynamics simulations. CHT was found to bind and sequester Aß peptides, thus limiting their ultimate aggregation numbers. The intensity of this inhibitory action was enhanced by CHT concentration, as well as CHT's pH-dependent degree of cationicity, corroborating experimental observations. Furthermore, CHT was found to reshape the conformational landscapes of Aß peptides, enriching collapsed peptides at near-physiological conditions of pH 7.5, and extended peptides at slightly acidic conditions of pH 6.5, where the charge profile of K16LVFFAE22 peptides remained unchanged. These conformational changes were limited to peptides in direct contact in CHT, thus emphasizing the influence of local environments on Aß conformations. These findings add to basic knowledge of the aggregation behaviour of Aß peptides, and could potentially guide the development of advanced CHT-based materials for the treatment of Alzheimer's disease.

Folding and modulation of the helical conformation of Glycophorin A by point mutations.

Transmembrane helix folding and self-association play important roles in biological signaling and transportation pathways across biomembranes. With molecular simulations, studies to explore the structural biochemistry of this process have been limited to focusing on individual fragments of this process - either helix formation or dimerization. While at an atomistic resolution, it can be prohibitive to access long spatio-temporal scales, at the coarse grained (CG) level, current methods either employ additional constraints to prevent spontaneous unfolding or have a low resolution on sidechain beads that restricts the study of dimer disruption caused by mutations. To address these research gaps, in this work, we apply our recent, in-house developed CG model (ProMPT) to study the folding and dimerization of Glycophorin A (GpA) and its mutants in the presence of Dodecyl-phosphocholine (DPC) micelles. Our results first validate the two-stage model that folding and dimerization are independent events for transmembrane helices and found a positive correlation between helix folding and DPC-peptide contacts. The wild type (WT) GpA is observed to be a right-handed dimer with specific GxxxG contacts, which agrees with experimental findings. Specific point mutations reveal several features responsible for the structural stability of GpA. While the T87L mutant forms anti-parallel dimers due to an absence of T87 interhelical hydrogen bonds, a slight loss in helicity and a hinge-like feature at the GxxxG region develops for the G79L mutant. We note that the local changes in the hydrophobic environment, affected by the point mutation, contribute to the development of this helical bend. This work presents a holistic overview of the structural stability of GpA in a micellar environment, while taking secondary structural fluctuations into account. Moreover, it presents opportunities for applications of computationally efficient CG models to study conformational alterations of transmembrane proteins that have physiological relevance.

Emergence of allostery through reorganization of protein residue network architecture.

Despite more than a century of study, consensus on the molecular basis of allostery remains elusive. A comparison of allosteric and non-allosteric members of a protein family can shed light on this important regulatory mechanism, and the bacterial biotin protein ligases, which catalyze post-translational biotin addition, provide an ideal system for such comparison. While the Class I bacterial ligases only function as enzymes, the bifunctional Class II ligases use the same structural architecture for an additional transcription repression function. This additional function depends on allosterically activated homodimerization followed by DNA binding. In this work, we used experimental, computational network, and bioinformatics analyses to uncover distinguishing features that enable allostery in the Class II biotin protein ligases. Experimental studies of the Class II Escherichia coli protein indicate that catalytic site residues are critical for both catalysis and allostery. However, allostery also depends on amino acids that are more broadly distributed throughout the protein structure. Energy-based community network analysis of representative Class I and Class II proteins reveals distinct residue community architectures, interactions among the communities, and responses of the network to allosteric effector binding. Bioinformatics mutual information analyses of multiple sequence alignments indicate distinct networks of coevolving residues in the two protein families. The results support the role of divergent local residue community network structures both inside and outside of the conserved enzyme active site combined with distinct inter-community interactions as keys to the emergence of allostery in the Class II biotin protein ligases.

Recovery of enzyme structure and activity following rehydration from ionic liquid.

Long-term preservation of proteins at room temperature continues to be a major challenge. Towards using ionic liquids (ILs) to address this challenge, here we present a combination of experiments and simulations to investigate changes in lysozyme upon rehydration from IL mixtures using two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO4] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et2PO4]). Various spectroscopic experiments and molecular dynamics simulations are performed to ascertain the structure and activity of lysozyme. Circular dichroism spectroscopy confirms that lysozyme maintains its secondary structure upon rehydration, even after 295 days. Increasing the IL concentration decreases the activity of lysozyme and is ultimately quenched at sufficiently high IL concentrations, but the rehydration of lysozyme from high IL concentrations completely restores its activity. Such rehydration occurs in the most common lysozyme activity assay, but without careful attention, this effect on the IL concentration can be overlooked. From simulations we observe occupation of [EMIM+] ions near the vicinity of the active site and the ligand-lysozyme complex is less stable in the presence of ILs, which results in the reduction of lysozyme activity. Upon rehydration, fast leaving of [EMIM+] is observed and the availability of active site is restored. In addition, suppression of structural fluctuations is also observed when in high IL concentrations, which also explains the decrease of activity. This structure suppression is recovered after undergoing rehydration. The return of native protein structure and activity indicates that after rehydration lysozyme returns to its original state. Our results also suggest a simple route to protein recovery following extended storage.

Effects of applied surface-tension on membrane-assisted Aß aggregation.

Accumulation of protein-based (Aß) aggregates on cellular membranes with varying structural properties is commonly recognized as the key step in Alzheimer's pathogenesis. But experimental and computational challenges have made this biophysical characterization difficult. In particular, studies connecting biological membrane organization and Aß aggregation are limited. While experiments have suggested that an increased membrane curvature results in faster Aß peptide aggregation in the context of Alzheimer's disease, a mechanistic explanation for this relation is missing. In this work, we are leveraging molecular simulations with a physics-based coarse grained model to address and understand the relationships between curved cellular membranes and aggregation of a model template peptide Aß 16-22. In agreement with experimental results, our simulations also suggest a positive correlation between increased peptide aggregation and membrane curvature. More curved membranes have higher lipid packing defects that engage peptide hydrophobic groups and promote faster diffusion leading to peptide fibrillar structures. In addition, we curated the effects of peptide aggregation on the membrane's structure and organization. Interfacial peptide aggregation results in heterogeneous headgroup-peptide interactions and an induced crowding effect at the lipid headgroup region, leading to a more ordered headgroup region and disordered lipid-tails at the membrane core. This work presents a mechanistic and morphological overview of the relationships between the biomembrane local structure and organization, and Aß peptide aggregation.

Tuning Allostery through Integration of Disorder to Order with a Residue Network.

In allostery, a signal from one site in a protein is transmitted to a second site to alter its function. Due to its ubiquity in biology and the potential for its exploitation in drug and protein design, the molecular basis of allosteric communication continues to be the subject of intense research. Although allosterically coupled sites are frequently characterized by disorder, how communication between disordered segments occurs remains obscure. Allosteric activation of Escherichia coli BirA dimerization occurs via coupled distant disorder-to-order transitions. In this work, combined structural and computational studies reveal an extensive residue network in BirA. Substitution of several network residues yields large perturbations to allostery. Force distribution analysis reveals that disruptions to the disorder-to-order transitions through amino acid substitution are manifested in shifts in the energy experienced by network residues as well as alterations in packing of an α-helix that plays a critical role in allostery. The combined results reveal a highly distributed allosteric mechanism that is robust to sequence change.

Dual mechanism of ionic liquid-induced protein unfolding.

Ionic liquids (ILs) are gaining attention as protein stabilizers and refolding additives. However, varying degrees of success with this approach motivates the need to better understand fundamental IL-protein interactions. A combination of experiment and simulation is used to investigate the thermal unfolding of lysozyme in the presence of two imidazolium-based ILs (1-ethyl-3-methylimidazolium ethylsulfate, [EMIM][EtSO4] and 1-ethyl-3-methylimidazolium diethylphosphate, [EMIM][Et2PO4]). Both ILs reduce lysozyme melting temperature Tm, but more gradually than strong denaturants. [EMIM][Et2PO4] lowers lysozyme Tm more readily than [EMIM][EtSO4], as well as requiring less energy to unfold the protein, as determined by the calorimetric enthalpy ΔH. Intrinsic fluorescence measurements indicate that both ILs bind to tryptophan residues in a dynamic mode, and furthermore, molecular dynamics simulations show a high density of [EMIM]+ near lysozyme's Trp62 residue. For both ILs approximately half of the [EMIM]+ cations near Trp62 show perfect alignment of their respective rings. The [EMIM]+ cations, having a "local" effect in binding to tryptophan, likely perturb a critically important Arg-Trp-Arg bridge through favorable π-π and cation-π interactions. Simulations show that the anions, [EtSO4]- and [Et2PO4]-, interact in a "global" manner with lysozyme, due to this protein's strong net positive charge. The anions also determine the local distribution of ions surrounding the protein. [Et2PO4]- is found to have a closer first coordination shell around the protein and stronger Coulomb interactions with lysozyme than [EtSO4]-, which could explain why the former anion is more destabilizing. Patching of ILs to the protein surface is also observed, suggesting there is no universal IL solvent for proteins, and highlighting the complexity of the IL-protein environment.

Computational insights into lipid assisted peptide misfolding and aggregation in neurodegeneration.

Peptide misfolding and aberrant assembly in membranous micro-environments have been associated with numerous neurodegenerative diseases. The biomolecular mechanisms and biophysical implications of these amyloid membrane interactions have been under extensive research and can assist in understanding disease pathogenesis and potential development of rational therapeutics. But, the complex nature and diversity of biomolecular interactions, structural transitions, and dependence on local environmental conditions have made accurate microscopic characterization challenging. In this review, using cases of Alzheimer's disease (amyloid-beta peptide), Parkinson's disease (alpha-synuclein peptide) and Huntington's disease (huntingtin protein), we illustrate existing challenges in experimental investigations and summarize recent relevant numerical simulation studies into amyloidogenic peptide-membrane interactions. In addition we project directions for future in silico studies and discuss shortcomings of current computational approaches.

Pathways of amyloid-beta absorption and aggregation in a membranous environment.

Aggregation of misfolded oligomeric amyloid-beta (Aß) peptides on lipid membranes has been identified as a primary event in Alzheimer's pathogenesis. However, the structural and dynamical features of this membrane assisted Aß aggregation have not been well characterized. The microscopic characterization of dynamic molecular-level interactions in peptide aggregation pathways has been challenging both computationally and experimentally. In this work, we explore differential patterns of membrane-induced Aß 16-22 (K-L-V-F-F-A-E) aggregation from the microscopic perspective of molecular interactions. Physics-based coarse-grained molecular dynamics (CG-MD) simulations were employed to investigate the effect of lipid headgroup charge - zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: POPC) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine: POPS) - on Aß 16-22 peptide aggregation. Our analyses present an extensive overview of multiple pathways for peptide absorption and biomechanical forces governing peptide folding and aggregation. In agreement with experimental observations, anionic POPS molecules promote extended configurations in Aß peptides that contribute towards faster emergence of ordered ß-sheet-rich peptide assemblies compared to POPC, suggesting faster fibrillation. In addition, lower cumulative rates of peptide aggregation in POPS due to higher peptide-lipid interactions and slower lipid diffusion result in multiple distinct ordered peptide aggregates that can serve as nucleation seeds for subsequent Aß aggregation. This study provides an in-silico assessment of experimentally observed aggregation patterns, presents new morphological insights and highlights the importance of lipid headgroup chemistry in modulating the peptide absorption and aggregation process.

Superrepression through Altered Corepressor-Activated Protein:Protein Interactions.

Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirAwt, five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multistep repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism.

How Hydrophobic Hydration Destabilizes Surfactant Micelles at Low Temperature: A Coarse-Grained Simulation Study.

Micelles are self-assembled aggregates of amphiphilic surfactant molecules that are important in a variety of applications, including drug delivery, detergency, and catalysis. It is known that the micellization process is driven by the same physiochemical forces that drive protein folding, aggregation, and biological membrane self-assembly. Nevertheless, the molecular details of how micelle stability changes in water at low temperature are not fully clear. We develop and use a coarse-grained model to investigate how the interplay between nonionic surfactants and the surrounding water at the nanoscale affects the stability of micelles at high and low temperatures. Simulations of preformed C12E5 micelles in explicit water at a range of temperatures reveal the existence of two distinct surfactant conformations within the micelle, a bent structure and an extended structure, the latter being more prevalent at low temperature. Favorable interactions of the surfactant with more ordered solvation water stabilizes the extended configuration, allowing nanoscale wetting of the dry, hydrophobic core of the micelle, leading to the micelle breaking. Taken together, our coarse-grained simulations unravel how energetic and structural changes of the surfactant and the surrounding water destabilize micelles at low temperature, which is a direct consequence of the weakened hydrophobicity. Our approach thus provides an effective mean for extracting the molecular-level changes during hydrophobicity-driven destabilization of molecular self-assembly, which is important in a wide range of fields, including biology, polymer science, and nanotechnology.

Mesoporous Encapsulated Chiral Nanogold for Use in Enantioselective Reactions.

Although various nanomaterials have been designed for biocatalysis, few of them can accelerate chemical reactions with high selectivity and stereocontrol, which remains them from being perfect alternatives to nature enzymes. Herein, inspired by the natural enzymes, an enantioselective nanomaterial has been constructed, with gold nanoparticles (AuNPs) as active centers, chiral cysteine (Cys) as selectors for chiral recognition, and expanded mesoporous silica (EMSN) as a skeleton of the artificial enzyme. In the oxidation of chiral 3,4-dihydroxy-phenylalanine (DOPA), the nanozyme with d-Cys shows preference to l-DOPA while the artificial enzyme with l-Cys shows preference to d-DOPA. Subsequent calculation of apparent steady-state kinetic parameters and activation energies together with molecular dynamics (MD) simulations showed that the different affinity precipitated by hydrogen bonding formation between chiral Cys and DOPA is the origin of chiral selectivity.

Long Distance Modulation of Disorder-to-Order Transitions in Protein Allostery.

Elucidation of the molecular details of allosteric communication between distant sites in a protein is key to understanding and manipulating many biological regulatory processes. Although protein disorder is acknowledged to play an important thermodynamic role in allostery, the molecular mechanisms by which this disorder is harnessed for long distance communication are known for a limited number of systems. Transcription repression by the Escherichia coli biotin repressor, BirA, is allosterically activated by binding of the small molecule effector biotinoyl-5'-AMP. The effector acts by promoting BirA dimerization, which is a prerequisite for sequence-specific binding to the biotin biosynthetic operon operator sequence. A 30 Å distance separates the effector binding and dimerization surfaces in BirA, and previous studies indicate that allostery is mediated, in part, by disorder-to-order transitions on the two coupled sites. In this work, combined experimental and computational methods have been applied to investigate the molecular basis of allosteric communication in BirA. Double-mutant cycle analysis coupled with thermodynamic measurements indicates functional coupling between residues in disordered loops on the two distant surfaces. All atom molecular dynamics simulations reveal that this coupling occurs through long distance reciprocal modulation of the structure and dynamics of disorder-to-order transitions on the two surfaces.

Acetylation within the First 17 Residues of Huntingtin Exon 1 Alters Aggregation and Lipid Binding.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization. As there are three lysines within Nt(17), we evaluated the impact of lysine acetylation on htt aggregation in solution and on model lipid bilayers. Acetylation of htt-exon1(51Q) and synthetic truncated htt-exon 1 mimicking peptides (Nt(17)-Q35-P10-KK) was achieved using a selective covalent label, sulfo-N-hydroxysuccinimide (NHSA). With this treatment, all three lysine residues (K6, K9, and K15) in Nt(17) were significantly acetylated. N-terminal htt acetylation retarded fibril formation in solution and promoted the formation of larger globular aggregates. Acetylated htt also bound lipid membranes and disrupted the lipid bilayer morphology less aggressively compared with the wild-type. Computational studies provided mechanistic insights into how acetylation alters the interaction of Nt(17) with lipid membranes. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers.