Biology subjects

Shirts, M. R.

Publications and source records attributed to Shirts, M. R..

10 recordsLinked to original sources

bioRxiv2026

Context-dependent peptide recognition shapes tyrosine kinase substrate specificity beyond consensus motifs

Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their sensitivity to sequence variation remains poorly understood. Here, we used microbial biosensors for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could confer sensitivity to substrate length or enhance selectivity for one PTK over others. Using a secondary decoy screen, we found sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. Our findings show how context-specific sequence features alter PTK substrate specificity far beyond what might be expected from classical consensus models and establish an experimental framework for defining the limits of substrate overlap between closely related kinases.

biochemistry↗

bioRxiv2026

A Hidden Binding Pocket in the β- ketoacyl-ACP Synthase FabB

Assembly-line enzymes carry out multi-step synthesis of diverse metabolites by using a handful of catalytic motifs in which minor structural differences control substrate specificity and reaction order. Here we examine differences in substrate binding to FabB and FabF, the two {beta}-ketoacyl-ACP synthases (KSs) responsible for fatty acid elongation in Escherichia coli, by exploring a peculiar mutational effect. In FabB, a blocking mutation in the acyl binding pocket yields a shifted, but broad product profile, while in FabF, the same mutation disrupts the binding of acyl chains longer than eight carbons (C8). X-ray crystal structures of the FabB mutant provide an explanation: a second, previously unobserved binding pocket allows medium-to-long acyl chains ([≥] C8) to bind with an alternate conformation. Molecular simulations suggest that this pocket is more stable in FabB than in FabF, where mutations reduce the catalytic competency of longer chains instead of shifting them to the alternate pocket. Our findings indicate that homologous KSs differ not only in their primary binding sites but also in the availability of alternative binding modes that can buffer against mutational effects and enable functional diversification.

biochemistry↗

bioRxiv2025

Computational design of dynamic biosensors for emerging synthetic opioids

Nitazenes are an emergent class of synthetic opioids that rival or exceed fentanyl in their potency. These compounds have been detected internationally in illicit drugs and are the cause of increasing numbers of hospitalizations and overdoses. New analogs are consistently released, making detection challenging - new ways of testing a wide range of nitazenes and their metabolic products are urgently needed. Here, we developed a computational protocol to redesign the plant abscisic acid receptor PYR1 to bind diverse nitazenes and maintain its dynamic transduction mechanism. The best design has a low nanomolar limit of detection in vitro against nitazene and menitazene. Deep mutational scanning yielded sensors able to recognize a range of clinically relevant nitazenes and the common metabolic byproduct in complex biological matrices with limited cross-specificity against unrelated opioids. Application of protein design tools on privileged receptors like PYR1 may yield general sensors for a wide range of applications in vitro and in vivo.

biochemistry↗

bioRxiv2025

Nonspecific interactions can lead to liquid-liquid phase separation in coiled-coil proteins models

Liquid-liquid phase separation (LLPS) is one mechanism that cells can use to organize biomolecules spatially and functionally. Some coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin and spd-5, are thought to LLPS, but it is currently unknown what parts of these proteins facilitate the process. It is thought, however, that the numerous CC domains in these proteins might be contributing to their LLPS. We recently showed, using computational studies and designed proteins, that CC domains can facilitate LLPS through specific interactions between the CC domains themselves, meaning that each CC was designed to interact only with a subset of other CCs in the system. This is in contrast to nonspecific interactions, where all CCs would be able to interact with all other CCs in the system, which is akin to some interactions (e.g.{pi} -{pi}) seen in phase-separating intrinsically disordered proteins. Because the specificity of interactions between natural CC domains is tunable in a sequence-dependent fashion, CC domains present a unique system that allows us to investigate the contributions of specific versus nonspecific interactions on LLPS. We show, in our computational system, that CC proteins with nonspecific interactions can LLPS but with less propensity compared to specific interactions. The LLPS propensity of CC proteins with nonspecific interactions can be improved by altering the structure and dynamics of linker segments, without directly changing the specificity of interactions. We also demonstrate that the number of intra-chain CC contacts plays a direct role in determining LLPS for nonspecifically interacting proteins. These results have broad implications for the role of linker segments--protein features beyond the interaction domains e.g. stickers--in protein LLPS and the formation of biomolecular condensates. STATEMENT OF SIGNIFICANCEModel coiled-coil proteins, which use coiled-coil domains as stickers, are capable of phase separation in a regime where intra-protein contacts interfere with the interactions which support phase separation. We explore ways to increase phase separation propensity without changing interaction specificity and find that the structure and size of spacers impacts LLPS propensity by affecting the formation of intra-chain interactions. This work demonstrates that protein LLPS might be controllable without directly affecting the cohesive parts of a protein i.e. stickers. This work also suggests that LLPS propensity might be a broadly accessible phenomenon for coiled-coil proteins.

biophysics↗

bioRxiv2024

Rationalizing diverse binding mechanisms to the same protein fold: in-sights for ligand recognition and biosensor design

The engineering of novel protein-ligand binding interactions, particularly for complex drug-like molecules, is an unsolved problem which could enable many practical applications of protein biosensors. In this work, we analyzed two engineer ed biosensors, derived from the plant hormone sensor PYR1, to recognize either the agrochemical mandipropamid or the synthetic cannabinoid WIN55,212-2. Using a combination of quantitative deep mutational scanning experiments and molecular dynamics simulations, we demonstrated that mutations at common positions can promote protein-ligand shape complementarity and revealed prominent differences in the electrostatic networks needed to complement diverse ligands. MD simulations indicate that both PYR1 protein-ligand complexes bind a single conformer of their target ligand that is close to the lowest free energy conformer. Computational design using a fixed conformer and rigid body orientation led to new WIN55,212-2 sensors with nanomolar limits of detection. This work reveals mechanisms by which the versatile PYR1 biosensor scaffold can bind diverse ligands. This work also provides computational methods to sample realistic ligand conformers and rigid body alignments that simplify the computational design of biosensors for novel ligands of interest.

bioengineering↗

bioRxiv2023

Coiled-coil domains are sufficient to drive liquid-liquid phase separation of proteins in molecular models

Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions which support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and has implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins. STATEMENT OF SIGNIFICANCEThe liquid-liquid phase separation of coiled-coil proteins has been implicated in the formation of membraneless organelles such as the centrosome and central spindle. Little is known about the features of these proteins that might drive their phase separation. We developed a modeling framework to investigate the potential role of coiled-coil domains in phase separation, and show that these domains are sufficient to drive the phenomenon in simulation. We additionally show the importance of multimerization state on the ability for such proteins to phase separate. This work suggests that coiled-coil domains should be considered for their contribution to protein phase separation.

biophysics↗

bioRxiv2023

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and 3 and 7 helices--the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites--an site and a {beta} site--can disrupt the allosteric network. Interestingly, binding to the site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the site, the terpenoids bind to the {beta} site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring--yet functionally similar--allosteric sites can have very different implications for inhibitor selectivity.

bioengineering↗

bioRxiv2019

The SAMPL6 SAMPLing challenge: Assessing the reliability and efficiency of binding free energy calculations

Approaches for computing small molecule binding free energies based on molecular simulations are now regularly being employed by academic and industry practitioners to study receptor-ligand systems and prioritize the synthesis of small molecules for ligand design. Given the variety of methods and implementations available, it is natural to ask how the convergence rates and final predictions of these methods compare. In this study, we describe the concept and results for the SAMPL6 SAMPLing challenge, the first challenge from the SAMPL series focusing on the assessment of convergence properties and reproducibility of binding free energy methodologies. We provided parameter files, partial charges, and multiple initial geometries for two octa-acid (OA) and one cucurbit[8]uril (CB8) host-guest systems. Participants submitted binding free energy predictions as a function of the number of force and energy evaluations for seven different alchemical and physical-pathway (i.e., potential of mean force and weighted ensemble of trajectories) methodologies implemented with the GROMACS, AMBER, NAMD, or OpenMM simulation engines. To rank the methods, we developed an efficiency statistic based on bias and variance of the free energy estimates. For the two small OA binders, the free energy estimates computed with alchemical and potential of mean force approaches show relatively similar variance and bias as a function of the number of energy/force evaluations, with the attach-pull-release (APR), GROMACS expanded ensemble, and NAMD double decoupling submissions obtaining the greatest efficiency. The differences between the methods increase when analyzing the CB8-quinine system, where both the guest size and correlation times for system dynamics are greater. For this system, nonequilibrium switching (GROMACS/NS-DS/SB) obtained the overall highest efficiency. Surprisingly, the results suggest that specifying force field parameters and partial charges is insufficient to generally ensure reproducibility, and we observe differences between seemingly converged predictions ranging approximately from 0.3 to 1.0 kcal/mol, even with almost identical simulations parameters and system setup (e.g., Lennard-Jones cutoff, ionic composition). Further work will be required to completely identify the exact source of these discrepancies. Among the conclusions emerging from the data, we found that Hamiltonian replica exchange--while displaying very small variance--can be affected by a slowly-decaying bias that depends on the initial population of the replicas, that bidirectional estimators are significantly more efficient than unidirectional estimators for nonequilibrium free energy calculations for systems considered, and that the Berendsen barostat introduces non-negligible artifacts in expanded ensemble simulations.

biophysics↗

bioRxiv2018

Open Force Field Consortium: Escaping atom types using direct chemical perception with SMIRNOFF v0.1

Here, we focus on testing and improving force fields for molecular modeling, which see widespread use in diverse areas of computational chemistry and biomolecular simulation. A key issue affecting the accuracy and transferrability of these force fields is the use of atom typing. Traditional approaches to defining molecular mechanics force fields must encode, within a discrete set of atom types, all information which will ever be needed about the chemical environment; parameters are then assigned by looking up combinations of these atom types in tables. This atom typing approach leads to a wide variety of problems such as inextensible atom-typing machinery, enormous difficulty in expanding parameters encoded by atom types, and unnecessarily proliferation of encoded parameters. Here, we describe a new approach to assigning parameters for molecular mechanics force fields based on the industry standard SMARTS chemical perception language (with extensions to identify specific atoms available in SMIRKS). In this approach, each force field term (bonds, angles, and torsions, and nonbonded interactions) features separate definitions assigned in a hierarchical manner without using atom types. We accomplish this using direct chemical perception, where parameters are assigned directly based on substructure queries operating on the molecule(s) being parameterized, thereby avoiding the intermediate step of assigning atom types -- a step which can be considered indirect chemical perception. Direct chemical perception allows for substantial simplification of force fields, as well as additional generality in the substructure queries. This approach is applicable to a wide variety of (bio)molecular systems, and can greatly reduce the number of parameters needed to create a complete force field. Further flexibility can also be gained by allowing force field terms to be interpolated based on the assignment of fractional bond orders via the same procedure used to assign partial charges. As an example of the utility of this approach, we provide a minimalist small molecule force field derived from Mercks parm@Frosst (an Amber parm99 descendant), in which a parameter definition file only {approx} 300 lines long can parameterize a large and diverse spectrum of pharmaceutically relevant small molecule chemical space. We benchmark this minimalist force field on the FreeSolv small molecule hydration free energy set and calculations of densities and dielectric constants from the ThermoML Archive, demonstrating that it achieves comparable accuracy to the Generalized Amber Force Field (GAFF) that consists of many thousands of parameters.

biophysics↗

bioRxiv2017

Approaches for calculating solvation free energies and enthalpies demonstrated with an update of the FreeSolv database

Solvation free energies can now be calculated precisely from molecular simulations, providing a valuable test of the energy functions underlying these simulations. Here, we briefly review \"alchemical\" approaches for calculating the solvation free energies of small, neutral organic molecules from molecular simulations, and illustrate by applying them to calculate aqueous solvation free energies (hydration free energies). These approaches use a non-physical pathway to compute free energy differences from a simulation or set of simulations and appear to be a particularly robust and general-purpose approach for this task. We also present an update (version 0.5) to our FreeSolv database of experimental and calculated hydration free energies of neutral compounds and provide input files in formats for several simulation packages. This revision to FreeSolv provides calculated values generated with a single protocol and software version, rather than the heterogeneous protocols used in the prior version of the database. We also further update the database to provide calculated enthalpies and entropies of hydration and some experimental enthalpies and entropies, as well as electrostatic and nonpolar components of solvation free energies.

pharmacology and toxicology↗