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bioRxiv2023

Single-molecule digital sizing of proteins in solution

Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable calibration-free single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

biophysics↗

bioRxiv2023

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects its membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/548826v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@23430org.highwire.dtl.DTLVardef@b06145org.highwire.dtl.DTLVardef@1c22579org.highwire.dtl.DTLVardef@12a56eb_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Lipidating the therapeutic peptide salmon calcitonin alters its biophysical characteristics, including oligomer size, hydrophobicity and membrane activity. - Fluorescent microscopy of single GUVs enables the determination of peptide mediated reporter dye influx behavior as either graded or All-or-None, which is coupled to either smaller membrane perturbations or peptide pore formation. - Modulating the number of hydrocarbons constituting the lipidation moieties determines the membrane permeation mechanism. - By increasing the lipid chain length lipidated of salmon calcitonin goes from displaying smaller membrane perturbations to a peptide pore formation mechanism. - Effective membrane translocation of lipidated salmon calcitonin requires a peptide mediated pore forming mechanism.

biophysics↗

bioRxiv2023

Phosphorylation modulates estrogen receptor disorder by altering long-range hydrophobic interactions

Protein intrinsic disorder is coupled to a range of biological phenomena, from gene regulation to cancer progression. Phosphorylation of the estrogen receptor (ER) at Ser118 through its disordered N-terminal domain (NTD) activates its transcriptional function, but it is challenging to rationalize how this modification regulates ER activity. Using biophysical approaches of small-angle X-ray scattering and nuclear magnetic resonance spectroscopy, we demonstrate that Ser118 phosphorylation triggers long-range conformational changes in ER-NTD, particularly between two hydrophobic clusters of residual structures. Alanine substitution of hydrophobic amino acids near Ser118 produces similar conformational alterations and rescues impaired ER activity caused by a phosphorylation-deficient mutant. These findings establish a direct link between phosphorylation-induced conformational changes and the activation function of this disordered protein as a promising avenue to block ER transcriptional activation.

biophysics↗

bioRxiv2023

Methods for making and observing model lipid droplets

The mechanisms by which the lipid droplet (LD) membrane is remodeled in concert with the activation of lipolysis incorporate a complex interplay of proteins, phospholipids, and neutral lipids. Model LDs (mLDs) provide an isolated, purified system for testing the mechanisms by which the droplet composition, size, shape, and tension affects triglyceride metabolism. Described here are methods of making and testing mLDs ranging from 0.1 to 40 {micro}m diameter with known composition. Methods are described for imaging mLDs with high-resolution microscopy during buffer exchanges for the measurement of membrane binding, diffusion, and tension via fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), atomic force microscopy (AFM), pendant droplet tensiometry, and imaging flow cytometry. These complementary, cross-validating methods of measuring LD membrane behavior reveal the interplay of biophysical processes in triglyceride metabolism.

biophysics↗

bioRxiv2023

Transcription regulates the spatio-temporal dynamics of genes through micro-compartmentalization

Although our understanding of the involvement of heterochromatin architectural factors in shaping nuclear organization is improving, there is still ongoing debate regarding the role of active genes in this process. In this study, we utilize publicly-available Micro-C data from mouse embryonic stem cells to investigate the relationship between gene transcription and 3D gene folding. Our analysis uncovers a nonmonotonic - globally positive - correlation between intragenic contact density and Pol II occupancy, independent of cohesin-based loop extrusion. Through the development of a biophysical model integrating the role of transcription dynamics within a polymer model of chromosome organization, we demonstrate that Pol II-mediated attractive interactions with limited valency between transcribed regions yield quantitative predictions consistent with chromosome-conformation-capture and live-imaging experiments. Our work provides compelling evidence that transcriptional activity shapes the 4D genome through Pol II-mediated micro-compartmentalization.

biophysics↗

bioRxiv2023

Unraveling membrane protein localization and stabilization in nanodiscs

Nanodiscs are nanoscale structures consisting of a lipid bilayer surrounded by membrane scaffold proteins (MSPs). They are widely used in the study of membrane proteins (MPs) because they provide a stable lipid environment. However, the precise mechanism governing MP behavior within the nanodisc remains elusive. Here, we examined the cryo-EM structures of various MPs reconstituted in nanodiscs from an electron microscopy database (EMPIAR). By analyzing the heterogeneity and interactions in the nanodiscs, we found that MPs within nanodiscs display a distinct spatial preference toward the edges of the nanodisc shells. Furthermore, we observed that MPs can establish direct, amphipathic interactions with the MSPs, promoting protein stability. These interactions may induce a rearrangement of the MSP-MSP interactions, leading to the formation of MP-MSP interactions Collectively, our study provides structural and biophysical insights into how nanodiscs contribute to MP structural behavior and stability. SIGNIFICANCEBy thoroughly examining multiple deposited datasets of membrane proteins (MPs) reconstituted in nanodiscs, we have gathered compelling evidence that MPs exhibit a clear spatial inclination toward the periphery of the nanodisc shells. Moreover, we have observed that MPs establish direct and amphipathic interactions with membrane scaffold proteins (MSPs). These interactions have the potential to induce a rearrangement of the MSP-MSP interactions, consequently forming MP-MSP interactions. Through quantitative analysis, we have successfully characterized the significant role played by these interactions in ensuring the overall stability of the proteins.

biophysics↗

bioRxiv2023

Emergence of selectivity and specificity in a coarse-grained model of the nuclear pore complex with sequence-agnostic FG-Nups

The role of hydrophobicity of phenylalanine-glycine nucleoporins (FG-Nups) in determining transport of receptor-bound cargo across the nuclear pore complex (NPC) is investigated using Langevin dynamics simulations. A coarse-grained, minimal model of the NPC, comprising a cylindrical pore and hydrophobic-hydrophilic random copolymers for FG-Nups was employed. Karyopherin-bound receptor-cargo complexes (Kaps) were modeled as rigid, coarse-grained spheres without (inert) and with (patchy) FG-binding hydrophobic domains. With a sequence-agnostic description of FG-Nups and the absence of any anisotropies associated with either NPC or cargo, the model described tracer transport only as a function of FG-Nup hydrophobicity, f. The simulations showed the emergence of two important features of cargo transport, namely, NPC selectivity and specificity. NPC selectivity to patchy tracers emerged due to hydrophobic Kap-FG interactions and despite the sequence-agnostic description of FG-Nups. Further, NPC selectivity was observed only in a specific range of FG-hydrophobic fraction, 0.05 [≤] f [≤] 0.20, resulting in specificity of NPC transport with respect to f. Significantly, this range corresponded to the number fraction of FG-repeats observed in both S. cerevisiae and H. sapiens NPCs. This established the centrality of FG-hydrophobic fraction in determining NPC transport, and provided a biophysical basis for conservation of FG-Nup hydrophobic fraction across evolutionarily distant NPCs. Specificity in NPC transport emerged from the formation of a hydrogel-like network inside the pore with a characteristic mesh size dependent on f. This network rejected cargo for f > 0.2 based on size exclusion which resulted in an enhanced translocation probability for 0.05 [≤] f [≤] 0.20. Thus, the NPC model provides a template for designing synthetic, biomimetic nanopores for macromolecular separations with high degrees of selectivity and specificity.

biophysics↗

bioRxiv2023

Single-molecule Imaging of SWI/SNF Chromatin Remodelers Reveal Multi-modal and Cancer-mutant-specific Landscape of DNA-binding Dynamics

Chromatin remodeling, carried out by multi-subunit remodeler complexes, alleviates topological constraints posed by nucleosomes to regulate genome access. Although mutations in the SWI/SNF subfamily of remodelers are implicated in >20% of human cancers, how misregulation of their intranuclear dynamics could underpin cancer remains poorly understood. Combining single-molecule tracking and fluorescence correlation spectroscopy, we probed the live-cell dynamics of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), revealing temporally distinct modes characteristic of free and chromatin-associated diffusion and chromatin-binding. Quantifying residence times of the fully assembled remodeler complex further resolved one transient and two stable binding fractions. Moreover, super-resolved density mapping of single-molecule binding using a newly devised strategy, termed STAR, revealed heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events preferentially cluster, with particular enrichment of consecutive longer-lived stable binding. Importantly, we showed that the bromodomain plays a key role in modulating the enhancement of remodeler binding dynamics in a DNA-accessibility-dependent manner, but does not facilitate targeting to hyperacetylated chromatin. Finally, we compared the chromatin-binding dynamics of seven common BRG1 mutants implicated in various cancers across tumor types, and uncovered systematic alterations in residence time, binding frequency, fraction of time bound, targeting efficiency and number of binding "hotspots" unique to each point/truncation mutant. Collectively, our findings shed critical insight into the multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers to selectively modulate genome accessibility, and could potentially serve as quantitative, mutant-specific signatures for cancers associated with remodeling misregulation. SIGNIFICANCE STATEMENTUsing two complementary approaches, we performed, to our knowledge, the first single-molecule quantification of live-cell dynamics of the fully assembled human SWI/SNF remodeler complex by correlating three key common subunits, and uncovered distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner. Our super-resolved mapping of chromatin-binding also revealed intranuclear "hotspots" where remodelers bind repeatedly in nanometer-scale clusters, as a potential strategy to promote remodeling at these loci. By leveraging previously under-explored parameters, our findings revealed a broader and multi-modal landscape that regulates SWI/SNF-mediated remodeling dynamics in space and time, and established the biophysical basis for aberrant remodeler-chromatin interactions associated with seven mutants implicated in various cancers, which could potentially serve as their unique identifying yardsticks.

biophysics↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2023

Iterative Machine Learning for Classification and Discovery of Single-molecule Unfolding Trajectories from Force Spectroscopy Data

We report the application of machine learning techniques to accelerate classification and analysis of protein unfolding trajectories from force spectroscopy data. Using kernel methods, logistic regression and triplet loss, we developed a workflow called Forced Unfolding and Supervised Iterative Online (FUSION) where a user classifies a small number of repeatable unfolding patterns encoded as image data, and a machine is tasked with identifying similar images to classify the remaining data. We tested the workflow using two case studies on a multi-domain XMod-Dockerin/Cohesin complex, validating the approach first using synthetic data generated with a Monte Carlo algorithm, and then deploying the method on experimental atomic force spectroscopy data. FUSION efficiently separated traces that passed quality filters from unusable ones, classified curves with high accuracy, and identified unfolding pathways undetected by the user. This study demonstrates the potential of machine learning to accelerate data analysis, and generate new insights in protein biophysics.

biophysics↗

bioRxiv2023

The α-crystallin chaperones undergo a quasi-ordered co-aggregation process in response to saturating client interaction

Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The -crystallins, A-crystallin (Ac) and B-crystallin (Bc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in Ac and Bc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined Ac and Bc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric -crystallin species isolated from aged lens tissue. This mechanism appears consistent across both Ac and Bc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.

biophysics↗

bioRxiv2023

Conformational transitions of the HIV-1 Gag polyprotein upon multimerization and gRNA binding

During the HIV-1 assembly process, the Gag polyprotein multimerizes at the producer cell plasma membrane, resulting in the formation of spherical immature virus particles. Gag-gRNA interactions play a crucial role in the multimerization process, which is yet to be fully understood. We have performed large-scale all-atom molecular dynamics simulations of membrane-bound full-length Gag dimer, hexamer, and 18-mer. The inter-domain dynamic correlation of Gag, quantified by the heterogeneous elastic network model (hENM) applied to the simulated trajectories, is observed to be altered by implicit gRNA binding, as well as the multimerization state of the Gag. The lateral dynamics of our simulated membrane-bound Gag proteins, with and without gRNA binding, agree with prior experimental data and help to validate our simulation models and methods. The gRNA binding is observed to impact mainly the SP1 domain of the 18-mer and the MA-CA linker domain of the hexamer. In the absence of gRNA binding, the independent dynamical motion of the NC domain results in a collapsed state of the dimeric Gag. Unlike stable SP1 helices in the six-helix bundle, without IP6 binding, the SP1 domain undergoes a spontaneous helix-to-coil transition in the dimeric Gag. Together, our findings reveal conformational switches of Gag at different stages of the multimerization process and predict that the gRNA binding reinforces an efficient binding surface of Gag for multimerization, as well as regulates the dynamic organization of the local membrane region itself. SignificanceGag(Pr55Gag) polyprotein orchestrates many essential events in HIV-1 assembly, including packaging of the genomic RNA (gRNA) in the immature virion. Although various experimental techniques, such as cryo-ET, X-ray, and NMR, have revealed structural properties of individual domains in the immature Gag clusters, structural and biophysical characterization of a full-length Gag molecule remains a challenge for existing experimental techniques. Using atomistic molecular dynamics simulations of the different model systems of Gag polyprotein, we present here a detailed structural characterization of Gag molecules in different multimerization states and interrogate the synergy between Gag-Gag, Gag-membrane, and Gag-gRNA interactions during the viral assembly process.

biophysics↗

bioRxiv2023

α-Synuclein emulsifies TDP-43 prion-like domain - RNA liquid droplets to promote heterotypic amyloid fibrils

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and -synuclein (S) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and S synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear whether and how S modulates TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale spatially-resolved spectroscopy, and other biophysical analyses, we uncover the interactions of S with phase-separated droplets. We learn that S acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets and emulsifying them. The hardening of the droplets that follow by S aggregates on the periphery, nucleates the formation of heterotypic TDP-43PrLD amyloid fibrils with structures distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of S as a Pickering agent in interacting with SGs and unmask the hitherto unknown role of S in modulating TDP-43 proteinopathies.

biophysics↗

bioRxiv2023

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.

biophysics↗

bioRxiv2023

Onsager's variational principle in proliferating biological tissues, in presenceof activity and anisotropy

A hallmark of biological cells is their ability to proliferate and of tissues their ability to grow. This is common in morphogenesis and embryogenesis but also in pathological conditions such as tumour growth. To consider these tissues from a physical point of view, it is necessary to derive fundamental relationships, in particular for velocities and density components, taking into account growth terms, chemical factors and the symmetry of cells and tissues. The aim is then to develop a consistent coarse-grained approach to these complex systems, which exhibit proliferation, disorder, anisotropy and activity at small scales. To this end, Onsagers variational principle allows the systematic derivation of flux-force relations in systems out of equilibrium and the principle of the extremum of dissipation, first formulated by Rayleigh and revisited by Onsager, finally leads to a consistent formulation for a continuous approach in terms of a coupled set of partial differential equations. Considering the growth and death rates as fluxes, as well as the chemical reactions driving the cellular activities, we derive the momentum equations based on a leading order physical expansion. Furthermore, we illustrate the different interactions for systems with nematic or polar order at small scales, and numerically solve the resulting system of partial differential equations in relevant biophysical growth examples. To conclude, we show that Onsagers variational principle is useful for systematically exploring the different scenarios in proliferating systems, and how morphogenesis depends on these interactions.

biophysics↗

bioRxiv2023

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes

The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

biophysics↗

bioRxiv2023

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated Protein Kinase C regulation

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms and {beta}II via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of Pin1 complexed to the C-terminal tail of PKC{beta}II reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a compact conformation in which it engages two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, the latter being a non-canonical Pin1-interacting element. The structural information, combined with the results of extensive binding studies and in vivo experiments suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates. Impact statementIntegrated biophysical, structural, and in vivo approaches demonstrate a non-canonical and non-isomerizable binding motif-dependent mode of protein kinase C regulation by the peptidyl-prolyl isomerase Pin1 in mammalian cells.

biophysics↗

bioRxiv2023

Structure of RADX and mechanism for regulation of RAD51 nucleofilaments

Replication fork reversal is a fundamental process required for resolution of encounters with DNA damage. A key step in the stabilization and eventual resolution of reversed forks is formation of RAD51 nucleoprotein filaments on exposed ssDNA. To avoid genome instability, RAD51 filaments are tightly controlled by a variety of positive and negative regulators. RADX is a recently discovered negative regulator that binds tightly to ssDNA, directly interacts with RAD51, and regulates replication fork reversal and stabilization in a context-dependent manner. Here we present a structure-based investigation of RADXs mechanism of action. Mass photometry experiments showed that RADX forms multiple oligomeric states in a concentration dependent manner, with a predominance of trimers in the presence of ssDNA. The structure of RADX, which has no structurally characterized orthologs, was determined ab initio by cryo-electron microscopy (EM) from maps in the 2-3 [A] range. The structure reveals the molecular basis for RADX oligomerization and binding of ssDNA binding. The binding of RADX to RAD51 filaments was imaged by negative stain EM, which showed a RADX oligomer at the end of filaments. Based on these results, we propose a model in which RADX functions by capping and restricting the growing end of RAD51 filaments. SignificanceDespite the central role of RAD51 in DNA replication and repair processes, the mechanisms of action of its many modulators are poorly understood. Here we combine structural and biophysical data to determine how the negative regulator RADX functions. We show that RADX oligomerizes upon binding DNA, and caps RAD51 filaments at the ends to prevent extension. This work advances knowledge of how RAD51 filaments can be modulated to regulate replication fork reversal and maintain genomic stability.

biophysics↗