Biology subjects

Sotomayor, M.

Publications and source records attributed to Sotomayor, M..

18 recordsLinked to original sources

bioRxiv2026

Bayesian-Steered Structure Prediction of Mechanical Biomolecules Using Twisted Diffusion

Deep learning approaches have revolutionized protein structure prediction. These tools are trained using experimental data and recapitulate reported conformations, but there is great interest in predicting conformations that may be functionally relevant although experimentally underrepresented. Since many modern structure prediction tools use generative artificial intelligence diffusion models, we reframe the search for alternative molecular conformations as that of sampling from a diffusion distribution conditioned using any arbitrary Bayesian likelihood. We implement a twisted diffusion sampler in Boltz-2 to sample this conditioned distribution and demonstrate the utility of this approach, which does not require any additional training of the neural network, by implementing a diffusion analog of steered molecular dynamics simulations applied to mechanical systems. We can reproduce predicted stretched states of fragments of DNA, the muscle protein titin, and the inner-ear protocadherin-15 protein, as well as open states of the MscL ion channel consistent with experimental results. We expect that steered structure predictions will help sample underrepresented and non-equilibrium conformations for many macromolecular systems.

bioinformatics↗

bioRxiv2026

In-Situ ssDNA Isolation from dsDNA Sources as a Streamlined Pathway to DNA Origami Assembly and Testing

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@f7ada7org.highwire.dtl.DTLVardef@a9bab8org.highwire.dtl.DTLVardef@121b695org.highwire.dtl.DTLVardef@5a1dea_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

PySteMoDA: An Open Source Python Package for the Analysis of Steered Molecular Dynamics Simulations Data

Molecular dynamics simulation is a powerful computational technique used for predicting and understanding the dynamic behavior of biomolecular systems. Steered molecular dynamics (SMD) simulations enable the study of force-induced processes in biomolecules, effectively mimicking single-molecule force spectroscopy experiments probing protein unfolding and receptor-ligand unbinding. Given the stochastic nature of these mechanical events, accurately exploring the dynamic behavior of biomolecules and extracting accurate physical information requires several in-silico experiments. This includes performing many pulling simulations at different velocities or force loading rates. The large amount of data obtained from these simulation sets requires efficient automated data processing tools. We present PySteMoDA, a novel Python package with a user-friendly graphical interface specifically designed for constant-velocity SMD data analysis. The automated force peak detection methods reduce user bias, improve accuracy, and accelerate data analysis. The package also allows identification of residues involved in mechanical events through computation of the time-dependent mechanical work and correlation factors between residue pairs. This package not only addresses automated data processing in SMD simulations and accurate parameter extraction, but also significantly enhances accessibility and usability. Through PySteMoDA, users can efficiently analyze simulation data without the barrier of coding, facilitating a wider range of investigations and insights in the field of computational biochemistry and biophysics.

biophysics↗

bioRxiv2025

TMC1 and TMC2 are cholesterol-dependent scramblases that regulate membrane homeostasis in auditory hair cells

TMC1 and TMC2, the pore-forming subunits of the mechanoelectrical transduction (MET) complex in inner ear sensory hair cells, are essential for auditory and vestibular function. Pathogenic mutations in TMC1 are a leading cause of genetic hearing loss, but their underlying cellular mechanisms remain poorly understood. Here, we reveal that TMC1 and TMC2 are cholesterol-regulated lipid scramblases whose activity modulates plasma membrane asymmetry. Using reconstituted proteoliposomes and molecular dynamics simulations, we demonstrate that both proteins facilitate phospholipid translocation across membrane bilayers, a process tuned by cholesterol and enhanced by deafness-causing TMC1 mutations. We show that this scramblase activity correlates with TMC1-dependent externalization of phosphatidylserine and membrane blebbing in murine auditory hair cells, linking TMC1-dependent membrane homeostasis dysregulation to auditory sensory cell pathology. These findings identify TMCs as a novel family of lipid scramblases, advancing our understanding of MET complex biology and offering mechanistic insight into membrane-driven forms of hereditary deafness.

neuroscience↗

bioRxiv2025

Groove architecture controls lipid scrambling in simulations of protein and model systems

Lipid bilayers are essential to life as they surround most cells and membrane-bound organelles. The integrity and fate of cells depend on the asymmetric makeup of lipid bilayers with various membrane proteins regulating the lipid composition of a bilayers two leaflets. Lipids scramblases are one of the primary regulators of lipid asymmetry in bilayers, spontaneously transferring lipids between membrane leaflets. Members of the TMEM16, OSCA/TMEM63, and TMC families have been suggested to be lipid scramblases. Despite significant differences, these proteins share a common structural architecture that features a membrane-exposed groove. The "credit card" mechanism proposes that lipids switch leaflets by moving their polar head groups either inside (partially dry) or on the surface of (wet) membrane-exposed, open hydrophilic grooves. However, emerging evidence of closed-groove scrambling challenges this model. Given the sequence diversity of groove-lining amino acids in TMEM16, OSCA/TMEM63, and TMC proteins, we hypothesized that lipid scrambling is primarily determined by groove architecture. To test this hypothesis and the credit card mechanism, we used coarse-grained molecular dynamics simulations of experimental structures and AlphaFold-generated models of six different scramblases in closed and open states. In these simulations, we observed little scramblase activity in most closed-state configurations but robust scrambling by all open-state models. We then built simplified TMEM16-based scramblases with only three bead types uniformly set for solvent-facing, transmembrane, and groove regions. We used this and further simplified models to vary groove surface hydrophilicity, groove surface geometry, and groove architecture. Our models support the partially dry and wet credit card mechanisms and suggest that groove architecture plays a more important role in facilitating lipid scrambling than the detailed sequence of groove-lining amino acids. STATEMENT OF SIGNIFICANCEMany biological processes, including blood clotting and apoptosis, are triggered by exposure of specific lipids on the outer leaflet of a lipid bilayer. Spontaneous diffusion of lipids between leaflets is slow, but scramblases can speed up transbilayer lipid exchange, a key step in signaling cascades. Here we used coarse-grained simulations to isolate and quantify the key biophysical properties of scramblases that modulate lipid movement across the bilayer. We found that the architecture of scramblases membrane-exposed groove is key to their function. This can be used to generate hypotheses about disease-causing mutations in known scramblases, guide drug design, and suggest favorable properties for de novo designed scramblases.

biophysics↗

bioRxiv2024

Transmembrane channel-like 4 and 5 proteins at microvillar tips are potential ion channels and lipid scramblases

Microvilli--membrane bound actin protrusions on the surface of epithelial cells--are sites of critical processes including absorption, secretion, and adhesion. Increasing evidence suggests microvilli are mechanosensitive, but underlying molecules and mechanisms remain unknown. Here, we localize transmembrane channel-like proteins 4 and 5 (TMC4 and 5) and calcium and integrin binding protein 3 (CIB3) to microvillar tips in intestinal epithelial cells, near glycocalyx insertion sites. We find that TMC5 colocalizes with CIB3 in cultured cells and that a TMC5 fragment forms a complex with CIB3 in vitro. Homology and AlphaFold2 models reveal a putative ion permeation pathway in TMC4 and 5, and molecular dynamics simulations predict both proteins can conduct ions and perform lipid scrambling. These findings raise the possibility that TMC4 and 5 interact with CIB3 at microvillar tips to form a mechanosensitive complex, akin to TMC1 and 2, and CIB2 and 3, within the mechanotransduction channel complex at the tips of inner ear stereocilia.

cell biology↗

bioRxiv2024

In-Silico Analyses of Molecular Force Sensors for Mechanical Characterization of Biological Systems

Mechanical forces play key roles in biological processes such as cell migration and sensory perception. In recent years molecular force sensors have been developed as tools for in situ force measurements. Here we use all-atom steered molecular dynamics simulations to predict and study the relationship between design parameters and mechanical properties for three types of molecular force sensors commonly used in cellular biological research: two peptide-and one DNA-based. The peptide-based sensors consist of a pair of fluorescent proteins, which can undergo Forster resonance energy transfer (FRET), linked by spider silk (GPGGA)n or synthetic (GGSGGS)n disordered regions. The DNA-based sensor consists of two fluorophore-labeled strands of DNA that can be unzipped or sheared upon force application with a FRET signal as readout of dissociation. We simulated nine sensors, three of each kind. After equilibration, flexible peptide linkers of three different lengths were stretched by applying forces to their N-and C-terminal C atoms in opposite directions. Similarly, we equilibrated a DNA-based sensor and pulled on the phosphate atom of the terminal guanine of one strand and a selected phosphate atom on the other strand in the opposite direction. These simulations were performed at constant velocity (0.01 nm/ns - 10 nm/ns) and constant force (10 pN - 500 pN) for all versions of the sensors. Our results show how the force response of these sensors depends on their length, sequence, configuration and loading rate. Mechanistic insights gained from simulations analyses indicate that interpretation of experimental results should consider the influence of transient formation of secondary structure in peptide-based sensors and of overstretching in DNA-based sensors. These predictions can guide optimal fluorophore choice and facilitate the rational design of new sensors for use in protein, DNA, hybrid systems, and molecular devices. STATEMENT OF SIGNIFICANCEBiomolecular structures involved in various biological processes, including muscle function and sensory perception, generate, convey, and respond to mechanical forces. In-vivo accurate measurement of these forces is challenging but needed to understand biological function. Here we present a comprehensive computational analysis of three different types of molecular force sensors used to report pico-Newton level forces in biomolecular systems. Our atom-level simulation predictions provide mechanistic insight that can facilitate experimental data interpretation, selection of sensor design parameters, and the development of new force sensors tailored to specific applications and environments.

biophysics↗

bioRxiv2024

Elasticity and Thermal Stability are Key Determinants of Hearing Rescue by Mini-Protocadherin-15 Proteins

Protocadherin-15 is a core protein component of inner-ear hair-cell tip links pulling on transduction channels essential for hearing and balance. Protocadherin-15 defects can result in non-syndromic deafness or Usher syndrome type 1F (USH1F) with hearing loss, balance deficits, and progressive blindness. Three rationally engineered shortened versions of protocadherin-15 (mini-PCDH15s) amenable for gene therapy have been used to rescue function in USH1F mouse models. Two can successfully or partially rescue hearing, while another one fails. Here we show that despite varying levels of hearing rescue, all three mini-PCDH15 versions can rescue hair-cell mechanotransduction. Negative-stain electron microscopy shows that all three versions form dimers like the wild-type protein, while crystal structures of some engineered fragments show that these can properly fold and bind calcium ions essential for function. In contrast, simulations predict distinct elasticities and nano differential scanning fluorimetry shows differences in melting temperature measurements. Our data suggest that elasticity and thermal stability are key determinants of sustained hearing rescue by mini-PCDH15s.

bioengineering↗

bioRxiv2023

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

neuroscience↗

bioRxiv2022

Interpreting the Evolutionary Echoes of a Protein Complex Essential for Inner-Ear Mechanosensation

The sensory epithelium of the inner ear, found in all extant lineages of vertebrates, has been subjected to over 500 million years of evolution, resulting in the complex inner ear of modern vertebrates. Inner-ear adaptations are as diverse as the species in which they are found, and such unique anatomical variations have been well studied. However, the evolutionary details of the molecular machinery that are required for hearing are less well known. Two molecules that are essential for hearing in vertebrates are cadherin-23 and protocadherin-15, proteins whose interaction with one another acts as the focal point of force transmission when converting sound waves into electrical signals that the brain can interpret. This interaction exists in every lineage of vertebrates, but little is known about the structure or mechanical properties of these proteins in most non-mammalian lineages. Here, we use various techniques to characterize the evolution of this protein interaction. Results show how evolutionary sequence changes in this complex affect its biophysical properties both in simulations and experiments, with variations in interaction strength and dynamics among extant vertebrate lineages. Evolutionary simulations also characterize how the biophysical properties of the complex in turn constrain its evolution and provide a possible explanation for the increase in deafness-causing mutants observed in cadherin-23 relative to protocadherin-15. Together, these results suggest a general picture of tip-link evolution in which selection acted to modify the tip-link interface, while subsequent neutral evolution combined with varying degrees of purifying selection drove additional diversification in modern tetrapods.

evolutionary biology↗

bioRxiv2021

In Silico Electrophysiology of Inner-Ear Mechanotransduction Channel TMC1 Models

Inner-ear sensory hair cells convert mechanical stimuli from sound and head movements into electrical signals during mechanotransduction. Identification of all molecular components of the inner-ear mechanotransduction apparatus is ongoing; however, there is strong evidence that TMC1 and TMC2 are pore-forming subunits of the complex. We present molecular dynamics simulations that probe ion conduction of TMC1 models built based on two different structures of related TMEM16 proteins. Unlike most channels, the TMC1 models do not show a central pore. Instead, simulations of these models in a membrane environment at various voltages reveal a peripheral permeation pathway that is exposed to lipids and that shows cation permeation at rates comparable to those measured in hair cells. Furthermore, our analyses suggest that TMC1 gating mechanisms involve protein conformational changes and tension-induced lipid-mediated pore widening. These results provide insights into ion conduction and activation mechanisms of hair-cell mechanotransduction channels essential for hearing and balance.

biophysics↗

bioRxiv2021

Elastic versus Brittle Mechanical Responses Predicted for Dimeric Cadherin Complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated , {beta}, and {gamma} clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins, and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function. Statement of SignificanceMulticellular organisms rely on cellular adhesion to survive, and this adhesion is mediated by diverse sets of proteins that include cadherins responsible for organ assembly and tissue integrity maintenance. As parts of cell-cell junctions in epithelial and cardiac tissues, classical cadherins experience forces and must be mechanically robust. In contrast, clustered protocadherins are responsible for neuronal connectivity and are exposed to more subtle mechanical stimuli. We used simulations to study the mechanics of isolated cadherin complexes and found that classical cadherins exhibit a two-phased elastic response that might prevent loss of adhesion during mild mechanical stress. Conversely, we predict that clustered protocadherin complexes are brittle. Our results suggest that each set of cadherins has evolved to adopt distinct mechanical properties.

biophysics↗

bioRxiv2021

Collective Mechanical Responses of Cadherin-Based Adhesive Junctions as Predicted by Simulations

Cadherin-based adherens junctions and desmosomes help stabilize cell-cell contacts with additional function in mechano-signaling, while clustered protocadherin junctions are responsible for directing neuronal circuits assembly. Structural models for adherens junctions formed by epithelial cadherin (CDH1) proteins indicate that their long, curved ectodomains arrange to form a periodic, two-dimensional lattice stabilized by tip-to-tip trans interactions (across junction) and lateral cis contacts. Less is known about the exact architecture of desmosomes, but desmoglein (DSG) and desmocollin (DSC) cadherin proteins are also thought to form ordered junctions. In contrast, clustered protocadherin (PCDH) based cell-cell contacts in neuronal tissues are thought to be responsible for self-recognition and avoidance, and structural models for clustered PCDH junctions show a linear arrangement in which their long and straight ectodomains form antiparallel overlapped trans complexes. Here we report all-atom molecular dynamics simulations testing the mechanics of minimalistic adhesive junctions formed by CDH1, DSG2 coupled to DSC1, and PCDH{gamma}B4, with systems encompassing up to 3.7 million atoms. Simulations generally predict a favored shearing pathway for the adherens junction model and a two-phased elastic response to tensile forces for the adhesive adherens junction and the desmosome models. Complexes within these junctions first unbend at low tensile force and then become stiff to unbind without unfolding. However, cis interactions in both the CDH1 and DSG2-DSC1 systems dictate varied mechanical responses of individual dimers within the junctions. Conversely, the clustered protocadherin PCDH{gamma}B4 junction lacks a distinct two-phased elastic response. Instead, applied tensile force strains trans interactions directly as there is little unbending of monomers within the junction. Transient intermediates, influenced by new cis interactions, are observed after the main rupture event. We suggest that these collective, complex mechanical responses mediated by cis contacts facilitate distinct functions in robust cell-cell adhesion for classical cadherins and in self-avoidance signaling for clustered PCDHs. Statement of SignificanceProteins that mediate cell-cell contacts often form aggregates in vivo where the tight packing of monomers into junctions is relevant to their function. Members of the cadherin superfamily of glycoproteins form large complexes in which their long ectodomains interact to mediate cell-cell adhesion. Here, we employ simulations to elucidate complex mechanical responses of five junction systems in response to force. Our results offer atomistic insights into the behavior of these proteins in a crowded physiological context, suggesting that classical cadherin complexes in adherens junctions and desmosomes act as molecular shock absorbers with responses modulated by dynamic lateral contacts, while clustered protocadherins form brittle junctions that upon stretching and unbinding form transient interfaces suitable for their critical role in neuronal self-recognition.

biophysics↗

bioRxiv2020

SPECIES-DEPENDENT HETEROPHILIC AND HOMOPHILIC CADHERIN INTERACTIONS IN INTESTINAL INTERMICROVILLAR LINKS

Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal pattern maintained by intermicrovillar links formed by two non-classical members of the cadherin superfamily of calcium-dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here we present X-ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal adhesive units.

biochemistry↗

bioRxiv2019

Structural determinants of protocadherin-15 elasticity and function in inner-ear mechanotransduction

Protocadherin-15 (PCDH15), an atypical member of the cadherin superfamily, is essential for vertebrate hearing and its dysfunction has been associated with deafness and progressive blindness. The PCDH15 ectodomain, made of eleven extracellular cadherin (EC1-11) repeats and a membrane adjacent domain (MAD12), assembles as a parallel homodimer that interacts with cadherin-23 (CDH23) to form the tip link, a fine filament necessary for inner-ear mechanotransduction. Here we report X-ray crystal structures of a PCDH15 + CDH23 heterotetrameric complex and ten PCDH15 fragments that were used to build complete high-resolution models of the monomeric PCDH15 ectodomain. Using molecular dynamics (MD) simulations and validated crystal contacts we propose models for complete PCDH15 parallel homodimers and the tip-link bond. Steered MD simulations of these models predict their strength and suggest conditions in which a multimodal PCDH15 ectodomain can act as a stiff or soft gating spring. These results provide a detailed view of the first molecular steps in inner-ear sensory transduction.

biophysics↗

bioRxiv2018

An Adhesive Interface For The Non-Clustered δ1 Protocadherin-1 Involved In Respiratory Diseases

Cadherins form a large family of calcium-dependent adhesive proteins involved in morphogenesis, cell differentiation, and neuronal connectivity. Non-clustered {delta}1 protocadherins form a cadherin subgroup of proteins with seven extracellular cadherin (EC) repeats and cytoplasmic domains distinct from those of classical cadherins. The non-clustered {delta}1 protocadherins mediate homophilic adhesion and have been implicated in various diseases including asthma, autism, and cancer. Here we present X-ray crystal structures of Protocadherin-1 (PCDH1), a {delta}1-protocadherin member essential for New World hantavirus infection that is typically expressed in the brain, airway epithelium, skin keratinocytes, and lungs. The structures suggest a binding mode that involves antiparallel overlap of repeats EC1 to EC4. Mutagenesis combined with binding assays and biochemical experiments validated this mode of adhesion. Overall, these results reveal the molecular mechanism underlying adhesiveness of PCDH1 and {delta}1-protocadherins, also shedding light on PCDH1s role in maintaining airway epithelial integrity, the loss of which causes respiratory diseases.

biochemistry↗

bioRxiv2018

Interaction specificity of clustered protocadherins inferred from sequence covariation and structural analysis

Clustered protocadherins are a large family of paralogous proteins that play important roles in neuronal development. The more than 50 clustered protocadherin isoforms have remarkable homophilic specificity for interactions between cellular surfaces that is controlled by a large antiparallel dimer interface formed by the first four extracellular cadherin (EC) domains. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and go through binding and unbinding events, but together they form a stable complex through polyvalency. Using sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs in the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions.

biophysics↗

bioRxiv2018

A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15

The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the \"giant\" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.

biophysics↗