Biology subjects

Klevit, R. E.

Publications and source records attributed to Klevit, R. E..

16 recordsLinked to original sources

bioRxiv2026

Function within Disorder: Small heat shock proteins use different functional regions to chaperone tau aggregation

In numerous neurodegenerative diseases known collectively as tauopathies, the microtubule-associated protein tau forms fibrillar aggregates that are hallmarks of disease pathology. Tauopathies represent a substantial fraction of diseases associated with protein misfolding. Cellular chaperones known as small heat shock proteins (sHSPs) play a critical role in maintaining protein homeostasis by delaying the onset of protein aggregation. Two sHSPs, HSPB1 (Hsp27) and HSPB5 (B-crystallin), are constitutively expressed in brain and neurons. Here, we show that HSPB1 and HSPB5 delay tau aggregation in vitro through distinct mechanisms dictated by their disordered N-terminal regions (NTRs). HSPB1 inhibits tau aggregation under normal cellular conditions, whereas HSPB5 displays activity towards tau when activated by stress conditions such as pH acidosis. Using chimeric HSPB1/HSPB5 constructs in which small NTR subregions are swapped, we identify functional regions within the NTRs that modulate chaperone function for tau. The functional regions contain known sites of phosphorylation, suggesting that they are also control points that respond to cellular stress conditions. Our findings support an emerging model in which specific functional motifs within disordered regions of sHSPs govern activity and client engagement under normal and stress conditions. Broader AudienceIn many neurodegenerative diseases, the microtubule-associated protein tau forms fibrillar aggregates in the brain. Small heat shock proteins (sHSP) help prevent such aggregation, but their mechanisms of action remain enigmatic. We show HSPB1 and HSBP5, two sHSPs that are abundant and co-localize with tau, delay the onset of tau aggregation through distinct mechanisms. Each relies on specific small regions within their disordered N- terminal domains whose accessibility can be regulated by stress conditions and post- translational modifications.

biochemistry↗

bioRxiv2025

Disorder with consequence: Phosphorylation sites in HSPB5 yield distinct structural outcomes

HSPB5, a member of the small heat shock protein family, acts as a first responder to cellular stress. One proposed mechanism of stress activation is phosphorylation. HSPB5 is phosphorylated at three sites--serine residues at positions 19, 45, and 59--located within its disordered N-terminal region (NTR). The extent of phosphorylation of the different sites leads to different cellular outcomes. HSPB5 forms polydisperse oligomers, where the NTR regions can either be exposed to the solvent or buried within the oligomer, forming internal contacts. We assessed the effect of single and triple phospho-mimicry on HSPB5 oligomeric properties. Our findings indicate that single phosphorylation causes localized and subtle changes in oligomer size, subunit exchange, hydrogen-deuterium protection patterns, and ability to delay aggregation of a known eye lens client, {gamma}D-crystallin. In contrast, the triple phosphomimic shows substantial structural and functional alterations. We provide a rationale for the increased chaperone activity observed in the S45D phosphomimic. Taken together, our results offer structural insights into how different phosphorylation events lead to distinct cellular outcomes.

biophysics↗

bioRxiv2025

Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.

Zinc is an essential metal that supports diverse cellular functions. Zinc exerts its biological activity through protein binding, serving as catalytic cofactors and structural stabilizers of many enzymes, transcription factors, and ubiquitin E3 ligases, among others. Despite total cellular zinc concentrations reaching hundreds of micromolar, free zinc levels are tightly buffered. Elevated free zinc promotes mismetalation and protein aggregation. While zinc is redox-inert, its cysteine-based protein ligands are readily oxidized. Oxidative modification of cysteines leads to zinc dissociation and a rapid increase in free zinc. With [~]3000 proteins in the human zinc proteome, uncontrolled zinc release could be highly deleterious. Metallothioneins buffer zinc under basal conditions, but their re-synthesis following oxidative inactivation occurs on the scale of hours, raising the question of how free zinc is managed in the interim. Histidine, the second most prevalent zinc-coordinating residue, is resistant to oxidative modification. We characterized zinc binding by the small heat shock protein HSPB5 (B-crystallin), a cysteine-free, histidine-rich protein chaperone that responds to cellular stress and found: (1) HSPB5 binds zinc with high affinity and rapid reversibility; (2) zinc binding requires the disordered HSPB5 N-terminal region; (3) zinc binding increases HSPB5 disorder; and (4) prolonged zinc exposure promotes formation of assemblies of oligomers cross-bridged by zinc. We propose that HSPB5 has evolved specialized zinc-dependent properties distinct among human sHSPs, enabling it to function not only as a protein chaperone but also as a conditional zinc reservoir under oxidative stress.

biophysics↗

bioRxiv2025

De Novo Design of Miniprotein Inhibitors of Bacterial Adhesins

The rise of multidrug-resistant bacterial infections necessitates the discovery of novel antimicrobial strategies. Here, we show that protein design provides a generalizable means of generating new antimicrobials by neutralizing the function of bacterial adhesins, which are virulence factors critical in host-pathogen interactions. We de novo designed high-affinity miniprotein binders to FimH and Abp chaperone usher pili adhesins from uropathogenic Escherichia coli and Acinetobacter baumannii, respectively, which are implicated in mediating both uncomplicated and catheter-associated urinary tract infections (UTI) responsible for significant morbidity worldwide. The designed antagonists have high specificity and stability, disrupt bacterial recognition of host receptors, block biofilm formation, and are effective in treating and preventing murine models of uncomplicated and catheter- associated UTIs in vivo.

microbiology↗

bioRxiv2025

Ultra-slow conformational dynamics and catch bond formation of a Bacterial Adhesin revealed by a single-domain variant of FimH.

Bacterial adhesins such as FimH are critical for host colonization and persistence under the mechanical forces encountered at sites of infection such as the urinary tract. Despite decades of research, the molecular mechanisms by which FimH--a key virulence factor of Escherichia coli--regulates its binding through conformational switching remain incompletely understood. FimH operates across a range of conformations that includes low- (LAS), intermediate-, high-affinity (HAS) states-- and forms catch bonds which paradoxically strengthen under force. The allosteric pathways governing these transitions remain poorly defined due to experimental limitations that restrict understanding of key dynamic phenomena that underlie ligand-triggered conformational shifts and force-induced long-lived interactions. Such understanding is central to drug discovery efforts to target bacterial adhesion. Here we present a model system that fully recapitulates the conformational repertoire of FimH in the absence of its pilin domain. Our findings demonstrate that a single mutation in the lectin domain induces the LAS while allowing for ligand-binding induced conformational change to the HAS and catch bond formation, mirroring the behavior of the native FimH adhesin. We propose a dynamic allosteric mechanism that involves ultra-slow, low-frequency dynamics for the ability of FimH and the bacteria that express it, to sustain long-lived interactions with mannose under both static and force conditions. SignificanceUrinary tract infections (UTIs) are among the most common bacterial infections, and their initiation depends on the ability of uropathogenic Escherichia coli (UPEC) to adhere to bladder cells. The adhesion is mediated by FimH, a protein on the outside of UPEC that binds mannose-containing glycoprotein receptors and strengthens its grip under shear stress via a catch-bond mechanism. To investigate FimH function, we engineered a variant that can adopt both low- and high-affinity states of FimH and can form catch bonds. We discovered that FimH is governed by ultra-slow conformational dynamics that vary even among structurally similar states. These findings provide a mechanistic framework for developing anti-adhesive therapies that target FimH dynamics, offering a novel strategy to prevent and treat UTIs.

biochemistry↗

bioRxiv2025

A framework for automated multimodal HDX-MS analysis

We present pyHXExpress, a customizable codebase for automated high-throughput multimodal analysis of all spectra generated from HDX-MS experiments. The workflow was validated against a synthetic test dataset to test the fitting algorithms and to confirm the statistical outputs. We further establish a framework for the determination of multimodality throughout a protein system by rigorous evaluation of multimodal fits across all peptide spectra. We demonstrate this approach using entire protein datasets to detect multimodality, conformational heterogeneity, and characterize dynamics of small heat shock protein HSPB5 and two disease mutants.

biophysics↗

bioRxiv2024

Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins.

immunology↗

bioRxiv2024

Force transmission through the inner kinetochore is enhanced by centromeric DNA sequences

Previously, we reconstituted a minimal functional kinetochore from recombinant Saccharomyces cerevisiae proteins that was capable of transmitting force from dynamic microtubules to nucleosomes containing the centromere-specific histone variant Cse4 (Hamilton et al. 2020). This work revealed two paths of force transmission through the inner kinetochore: through Mif2 and through the Okp1/Ame1 complex (OA). Here, using a chimeric DNA sequence that contains crucial centromere-determining elements of the budding yeast point centromere, we demonstrate that the presence of centromeric DNA sequences in Cse4-containing nucleosomes significantly strengthens OA-mediated linkages. Our findings indicate that centromeric sequences are important for the transmission of microtubule-based forces to the chromosome.

biochemistry↗

bioRxiv2024

SUMO's intrinsically disordered N-terminus is an intramolecular inhibitor of SUMO - SIM interactions

Ubiquitin-related proteins of the SUMO family are reversibly attached to thousands of proteins in eukaryotic cells. Many SUMO substrates, effectors and enzymes carry short motifs (SIMs) that mediate low affinity interactions with SUMO proteins. How specificity is achieved in target selection, SUMO paralogue choice and SUMO-dependent interactions is largely unknown. A unique but poorly understood feature of SUMO proteins is their intrinsically disordered N-terminus. We reveal a function for N-termini of human, C. elegans, and yeast SUMO proteins as intramolecular inhibitors of SUMO- SIM interactions. Mutational analyses, NMR spectroscopy, and Molecular Dynamics simulations indicate that SUMOs N-terminus can inhibit SIM binding by fast and fuzzy interactions with SUMO s core. Deletion of the C. elegans SUMO1 N-terminus leads to p53-dependent apoptosis during germline development, indicating an important role in DNA damage repair. Our findings reveal a mechanism of disorder-based autoinhibition that contributes to the specificity of SUMOylation and SUMO-dependent interactions.

biochemistry↗

bioRxiv2022

Multivalent BARD1-nucleosome interactions facilitate H2A ubiquitylation by BRCA1/BARD1

BRCA1/BARD1 is a tumor suppressor E3 ubiquitin (Ub) ligase with roles in DNA damage repair and in transcriptional regulation. BRCA1/BARD1 RING domains interact with nucleosomes to facilitate mono-ubiquitylation of distinct residues on the C-terminal tail of histone H2A. These enzymatic domains constitute a small fraction of the heterodimer, raising the possibility of functional chromatin interactions involving other regions such as the BARD1 C-terminal domains that bind nucleosomes containing the DNA damage signal H2A K15-Ub and H4 K20me0, or portions of the expansive intrinsically disordered regions found in both subunits. Herein, we reveal novel interactions that support robust H2A ubiquitylation activity mediated through a high-affinity, intrinsically disordered DNA-binding region of BARD1. These interactions support BRCA1/BARD1 recruitment to chromatin and sites of DNA damage in cells and contribute to their survival. We also reveal distinct BRCA1/BARD1 complexes that depend on the presence of H2A K15-Ub, including a complex where a single BARD1 subunit spans adjacent nucleosome units. Our findings identify an extensive network of multivalent BARD1- nucleosome interactions that serve as a platform for BRCA1/BARD1-associated functions on chromatin.

biochemistry↗

bioRxiv2022

HSPB5 disease-associated mutations have long-range effects on structure and dynamics through networks of quasi-ordered interactions

Found from bacteria to humans, small heat shock proteins (sHSPs) are the least understood protein chaperones. HSPB5 (or B-crystallin) is among the most widely expressed of the ten human sHSPs, including in muscle, brain, and eye lens where it is constitutively present at very high levels and carries out a myriad of functions. A high content of disorder in HSPB5 has stymied efforts to uncover how its structure gives rise to function. To uncover its mechanisms of action, we compared human HSPB5 and two disease-associated mutants, R120G and D109H. Expecting to learn how the mutations lead to loss of function, we found instead that the mutants are constitutively activated chaperones while wild-type HSPB5 can transition reversibly between non-activated (low activity) and activated (high activity) states in response to changing conditions. Techniques that provide information regarding interactions and accessibility of disordered regions revealed that the disordered N-terminal regions (NTR) that are required for chaperone activity exist in a complicated interaction network within HSPB5 oligomers and are sequestered from solvent in non-activated states. Either mutation or an activating pH change cause rearrangements in the network that expose parts of the NTR, making them more available to bind an aggregating client. While beneficial in the short-term, failure of the mutants to adopt a state with lower activity and lower NTR accessibility leads to increased co-aggregation propensity and, presumably, early cataract. The results support a model where chaperone activity and solubility are modulated through the quasi-ordered NTR and its multiple competing interactions. SignificanceSmall heat shock proteins (sHSPs) are the oldest known protein chaperones, but how they recognize misfolding proteins in early stages of aggregation is unknown. Disordered regions within sHSPs are critical to their function, raising the question of how disorder recognizes disorder. We investigated a human sHSP (HSPB5) and two disease mutants using approaches that provide residue-level information in regions of disorder. The findings reveal that large, heterogeneous oligomers of HSPB5 contain complicated networks of interactions involving their disordered regions that protect them from solvent. Conditions associated with stress or disease mutations cause a rearrangement of the interaction network and enhancement of chaperone activity. The study provides new information regarding how disordered regions are modulated in a network of competing interactions.

biophysics↗

bioRxiv2019

Mediator subunit Med15 dictates the conserved 'fuzzy' binding mechanism of yeast transcription activators Gal4 and Gcn4

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in both transcription repression and activation through sequence-dependent binding to Gal80 repressor and sequence-independent binding to Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two ADs of different sequence, interact nearly identically with Med15. The findings that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts with Gal80 via a tight structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein interaction.

molecular biology↗

bioRxiv2019

Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of \"quasi-ordered\" states.

Small heat shock proteins (sHPSs) are natures \"first responders\" to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured -crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of \"quasi-ordered\" NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.

biochemistry↗

bioRxiv2019

Indirect sexual selection drives rapid sperm protein evolution

Sexual selection can explain rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if they are not directly involved in fertilization. Here we demonstrate that FITZAP, an intrinsically disordered sperm protein in the marine mollusk abalone, exploits differences in the intracellular and oceanic ionic environments to package the fertilization protein lysin at extraordinary concentrations inside sperm by forming Fuzzy Interacting Transient Zwitterion (FITZ) complexes. FITZAP binds lysin at the same protein interface as its egg receptor VERL, and as sexual selection rapidly alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. Consequently, FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution.\n\nOne Sentence SummaryStructural study of sperm proteins reveals a novel protein packaging/dispersion system embedded in a coevolutionary arms race.

biochemistry↗

bioRxiv2017

Transcription activator-coactivator specificity is mediated by a large and dynamic fuzzy protein-protein complex

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient activation by yeast Gcn4 requires tandem Gcn4 ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous, containing nearly all possible AD-ABD interactions. This complex forms using a dynamic fuzzy protein-protein interface where ADs use hydrophobic residues to bind hydrophobic surfaces of the ABDs in multiple orientations. This combinatorial mechanism allows individual interactions of low affinity and specificity to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators and allows great flexibility in combinations of activators that synergize to regulate genes with variable coactivator requirements.

molecular biology↗

bioRxiv2017

Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and VERL are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin, including crystal structures of multiple orthologs, it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (Haliotis rufescens). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N-and C-termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and / or accelerated evolution. These flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.\n\nSignificanceThe fertilization of eggs by sperm is a critical biological process for nearly all sexually reproducing organisms to propagate their genetic information. Despite the importance of fertilization, the molecules that mediate egg-sperm interactions have been characterized for only a few species, and the biochemical mechanisms underlying these interactions are even less well understood. In the marine mollusk abalone, sperm lysin interacts with egg VERL in a species-specific manner to facilitate fertilization. In this report, we characterized the solution structure and molecular evolution of sperm lysin from red abalone (Haliotis rufescens), and identified the VERL binding interface as well as important lysin dimer interactions that have emerged as part of an incessant sexual arms race.

evolutionary biology↗