Exchange Mass Spectrometry Research Articles

Hydrogen-Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE.

Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein-protein and protein-peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by size-exclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe4S4. Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2 O. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

Recognition of Aedes aegypti Mosquito Saliva Protein LTRIN by the Human Receptor LTβR for Controlling the Immune Response.

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.

Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation.

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-β component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.

Significance of Histidine Hydrogen-Deuterium Exchange Mass Spectrometry in Protein Structural Biology.

Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determining the acid-base dissociation constant (Ka) of histidine imidazole rings in proteins often yields valuable insights into protein functions. Significant efforts have been dedicated to measuring the pKa values of histidine residues in various proteins, with nuclear magnetic resonance (NMR) spectroscopy being the most commonly used technique. However, NMR-based methods encounter challenges in assigning signals to individual imidazole rings and require a substantial amount of proteins. To address these issues associated with NMR-based approaches, a mass-spectrometry-based method known as histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) has been developed. This technique not only determines the pKa values of histidine imidazole groups but also quantifies their solvent accessibility. His-HDX-MS has proven effective across diverse proteins, showcasing its utility. This review aims to clarify the fundamental principles of His-HDX-MS, detail the experimental workflow, explain data analysis procedures and provide guidance for interpreting the obtained results.

Automated high-throughput buffer exchange platform enhances rapid flow analysis of antibody drug conjugates by high resolution mass spectrometry

Antibody drug conjugates (ADCs) are an increasingly important therapeutic class of molecules for the treatment of cancer. Average drug-to-antibody ratio (DAR) and drug-load distribution are critical quality attributes of ADCs with the potential to impact efficacy and toxicity of the molecule and need to be analytically characterized and understood. Several platform methods including hydrophobic interaction chromatography (HIC) and native size-exclusion chromatography-mass spectrometry (nSEC-MS) have been developed for that purpose; however, each presents some limitations. In this work, we assessed a new sample preparation and buffer exchange platform coupled with high-resolution mass spectrometry for characterizing the drug-load and distribution of several cysteine-linked ADCs conjugated with a variety of chemotypes. Several criteria were evaluated during the optimization of the buffer exchange-mass spectrometry system performance and the data generated with the system were compared with results from nSEC-MS and HIC. The results indicated that the platform enables automated and high throughput quantitative DAR characterization for antibody-drug conjugates with high reproducibility and offers several key advantages over existing approaches that are used for chemotype-agnostic ADC characterization.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking β-sheets across the VHH protein, thereby preventing β-sheet-β-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

Unveiling the domain-specific and RAS isoform-specific details of BRAF kinase regulation.

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various types of cancer and mutant RAS, the upstream regulator of BRAF, is a driver of up to one-third of all cancers. BRAF interacts with RAS and undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer, which propagates downstream signaling. Because of BRAF's complex regulation mechanism, the exact order and magnitude of its activation steps have yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation through pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activation of BRAF in a RAS isoform-specific manner. Moreover, we quantified the binding affinities between BRAF N-terminal and kinase domains (KD) to reveal their individual roles in autoinhibition. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the N-terminal domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insight into the activation mechanism of BRAF kinase to guide the development of new therapeutic strategies for cancer treatment.

Unveiling the domain-specific and RAS isoform-specific details of BRAF kinase regulation

BRAF is a key member in the MAPK signaling pathway essential for cell growth, proliferation, and differentiation. Mutant BRAF is often the underlying cause of various types of cancer and mutant RAS, the upstream regulator of BRAF, is a driver of up to one-third of all cancers. BRAF interacts with RAS and undergoes a conformational change from an inactive, autoinhibited monomer to an active dimer, which propagates downstream signaling. Because of BRAF’s complex regulation mechanism, the exact order and magnitude of its activation steps have yet to be confirmed experimentally. By studying the inter- and intramolecular interactions of BRAF, we unveil the domain-specific and isoform-specific details of BRAF regulation through pulldown assays, open surface plasmon resonance (OpenSPR), and hydrogen-deuterium exchange mass spectrometry (HDX-MS). We demonstrate that the BRAF specific region (BSR) and cysteine rich domain (CRD) play a crucial role in regulating the activation of BRAF in a RAS isoform-specific manner. Moreover, we quantified the binding affinities between BRAF N-terminal and kinase domains (KD) to reveal their individual roles in autoinhibition. Our findings also indicate that oncogenic BRAF-KDD594G mutant has a lower affinity for the N-terminal domains, implicating that pathogenic BRAF acts through decreased propensity for autoinhibition. Collectively, our study provides valuable insight into the activation mechanism of BRAF kinase to guide the development of new therapeutic strategies for cancer treatment.

Structural flexibility and heterogeneity of recombinant human glial fibrillary acidic protein (GFAP).

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway.

To reduce the risk of resistance development, a novel fungicide with dual specificity is demanded. Trehalose is absent in animals, and its synthases, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), are safe fungicide targets. Here, we report the discovery of a dual-specificity inhibitor of MoTps1 (Magnaporthe oryzae Tps1, TPS) and MoTps2 (M. oryzae Tps2, TPP). The inhibitor, named A1-4, was obtained from a virtual screening and subsequent surface plasmon resonance screening. In in vitro assays, A1-4 interacts with MoTps1 and MoTps2-TPP (MoTps2 TPP domain) and inhibits their enzyme activities. In biological activity assays, A1-4 not only inhibits the virulence of M. oryzae on host but also causes aggregation of conidia cytosol, which is a characteristic phenotype of MoTps2. Furthermore, hydrogen/deuterium exchange mass spectrometry assays support the notion that A1-4 binds to the substrate pockets of TPS and TPP. Collectively, A1-4 is a promising hit compound for the development of safe fungicide with dual-target specificity.

Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T.gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an invivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T.gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T.gondii fitness.

Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 μM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Conformational plasticity of RAS Q61 family of neoepitopes results in distinct features for targeted recognition

The conformational landscapes of peptide/human leucocyte antigen (pHLA) protein complexes encompassing tumor neoantigens provide a rationale for target selection towards autologous T cell, vaccine, and antibody-based therapeutic modalities. Here, using complementary biophysical and computational methods, we characterize recurrent RAS55-64 Q61 neoepitopes presented by the common HLA-A*01:01 allotype. We integrate sparse NMR restraints with Rosetta docking to determine the solution structure of NRASQ61K/HLA-A*01:01, which enables modeling of other common RAS55-64 neoepitopes. Hydrogen/deuterium exchange mass spectrometry experiments alongside molecular dynamics simulations reveal differences in solvent accessibility and conformational plasticity across a panel of common Q61 neoepitopes that are relevant for recognition by immunoreceptors. Finally, we predict binding and provide structural models of NRASQ61K antigens spanning the entire HLA allelic landscape, together with in vitro validation for HLA-A*01:191, HLA-B*15:01, and HLA-C*08:02. Our work provides a basis to delineate the solution surface features and immunogenicity of clinically relevant neoepitope/HLA targets for cancer therapy.

Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Unravelling the mechanism of neurotensin recognition by neurotensin receptor 1

The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS1) using 19F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS1. Numerical analysis of the kinetic data of neurotensin binding to NTS1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs.

Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.

Deciphering the Roles of the Key Oligomerization Domains in Amyloid‐β to Design Amyloid Aggregation Inhibitors

AbstractBackgroundProtein folding issues are significant difficulties since they are linked to various diseases, including dementia. Fibrillar amyloid aggregates composed of misfolded proteins are pathological features of several neurodegenerative diseases. Numerous attempts have been made to treat neurodegenerative diseases by focusing on amyloid aggregates of pathologically disordered proteins. The first Alzheimer’s disease (AD) curative drug was introduced as a consequence of attempts to combat the most frequent kind of dementia. Even though antibody strategies reduce clinical deterioration in mild cognitive disease, alternate techniques must be developed over a single approach (i.e., antibody treatment). Recent studies have attempted to rationally design candidate drugs to improve existing therapeutic approaches or suppress the fibrillar amyloid aggregation process altogether. Prompted by previous findings, we rationally designed amyloid‐β (1‐42) point mutants to inhibit amyloid aggregation.MethodWe investigated the structural ensembles of the monomers using small‐angle X‐ray scattering. In addition, we performed hydrogen‐deuterium exchange mass spectrometry (MS) and ion mobility MS to elucidate the molecular principles governing Aβ42‐inhibitor interactions. We also used microsecond‐scale molecular dynamics simulations to investigate the structures and interactions, and combined them with electron transfer dissociation MS analysis to tackle challenges in identifying the multiple binding sites of intrinsically disordered protein complexes.ResultSpecifically designed mutations disrupt not only amyloid aggregation but reduce its cytotoxicity. Furthermore, we proposed minimal mutation sites to control amyloid‐β (1‐42) self‐assembly behavior. We are now determining the key domains of amyloid‐β (1‐42) that facilitate its self‐assembly to design aggregation inhibitors as potential therapeutic agents. We identified three critical hydrophobic domains (17LVF19, 32IGL34, and 41IA42) on amyloid‐β (1‐42) and investigated their role in the protein’s self‐assembly process. Our results suggest that interchain interactions in the central hydrophobic region (17LVF19) of amyloid‐β (1‐42) are essential for fibrillar aggregation and that their interaction with other domains is related to the accessibility of the central hydrophobic region for initiating the pathological cascade. Our findings shed light on the mechanisms underlying amyloid‐β (1‐42) self‐assembly and reveal key structural domains that aid in this process.ConclusionOur findings can be used to improve the rational design of amyloid‐β (1‐42) aggregation inhibitors in the future.