Biology subjects

Brooks, C. L.

Publications and source records attributed to Brooks, C. L..

8 recordsLinked to original sources

bioRxiv2026

Surface electrostatic networks control hydrophobic core remodeling in a pH-dependent switching protein

Communication between protein surfaces and their buried cores is central to protein structure and function, yet this phenomenon remains challenging to predict and control at high resolution. Changes in the protonation of surface ionizable residues communicate with the hydrophobic core, for example, in diverse pH-dependent protein functions. Hisactophilin, a histidine-rich actin- and membrane-binding protein, provides a general model for exploring such communication as it exhibits a finely tuned pH-regulated myristoyl-switching function. Upon reversible proton binding, the myristoyl group shifts between being sequestered in the hydrophobic core and more solvent accessible. In the current study we utilize experimental and computational approaches we uncover how binding of [~]1.5 net protons alters electrostatic interactions involving ionizable residues distributed across much of the protein surface. These changes are transmitted to the hydrophobic core through dynamic communities of ionizable and hydrophobic residues which substantially rearrange upon switching. The effects of mutating individual ionizable residues are weaker than those of core hydrophobic residues, and only combined mutation of multiple ionizable residues caused substantial functional change. Together, these results reveal how communication between surface ionizable residues and the hydrophobic core is mediated by extensive interaction networks that reorganize in response to changes in protonation. These results may provide general insights for understanding protein cooperativity and the coupling of surface and core residues in protein function, disease, evolution, engineering, and design. Significance StatementHow changes on the protein surface, such as proton binding to ionizable amino acids, are communicated to the protein core to regulate protein stability and function remains ill-defined. Synthesis of experimental and computational analyses resolves the distributed networks of surface ionizable residue interactions coupled to the hydrophobic core that control pH-dependent myristoyl switching in hisactophilin. Small changes in protonation that create and alleviate local electrostatic repulsion give rise to protein-wide changes in fluctuating surface-core interactions. This distributed electrostatics-core coupling mechanism may help explain the often underrecognized and long-range impacts of ionizable residues in proteins and provide a framework for interpreting the effects of mutations in fundamental and applied protein science.

biophysics↗

bioRxiv2026

FAIM Inhibits Insulin Amyloidogenesis through a Noncanonical Aggregation Pathway

Insulin can misfold and assemble into amyloid fibrils, a process linked not only to complications of insulin therapy but also to proteotoxic stress in pancreatic {beta}-cells. Despite growing interest in the pathological consequences of insulin aggregation, prevention efforts are limited by an incomplete understanding of the endogenous mechanisms that counteract it. Here, we identify Fas apoptosis inhibitory molecule (FAIM) as an endogenous suppressor of insulin amyloid formation. FAIM reduces {beta}-sheet formation and redirects insulin toward disordered, growth-incompetent assemblies. Further, FAIM attenuates the cytotoxicity of insulin aggregates in vitro. We hypothesize that this effect arises from masking aggregation-prone regions of insulin and show through structural modeling that FAIM interacts with both insulin chains. These findings extend the anti-aggregation function of FAIM to insulin and suggest a mechanism for endogenous suppression of insulin amyloid formation. More broadly, our results provide insight into the regulation of insulin assembly and highlight FAIM as a candidate modulator of proteostasis in metabolic disease. Statement for a broader audienceInsulin can clump together into harmful aggregates, contributing to complications of insulin therapy and potentially damaging the insulin-producing cells of the pancreas. This study identifies the naturally occurring protein FAIM as a protective factor that inhibits the formation of these harmful aggregates and reduces their toxicity. These findings improve our understanding of how cells protect insulin from harmful aggregation and may open new avenues for developing therapies to combat diabetes-related protein aggregation.

biochemistry↗

bioRxiv2024

Identifying Artifacts from Large Library Docking

While large library docking has discovered potent ligands for multiple targets, as the libraries have grown, the very top of the hit-lists can become populated with artifacts that cheat our scoring functions. Though these cheating molecules are rare, they become ever-more dominant with library growth. Here, we investigate rescoring top-ranked molecules from docking screens with orthogonal methods to identify these artifacts, exploring implicit solvent models and absolute binding free energy perturbation (AB-FEP) as cross-filters. In retrospective studies, this approach deprioritized high-ranking non-binders for nine targets while leaving true ligands relatively unaffected. We tested the method prospectively against results from large library docking AmpC {beta}-lactamase. From the very top of the docking hit lists, we prioritized 128 molecules for synthesis and experimental testing, a mixture of 39 molecules that rescoring flagged as likely cheaters and another 89 that were plausible true actives. None of the 39 predicted cheating compounds inhibited AmpC up to 200{micro}M in enzyme assays, while 57% of the 89 plausible true actives did do so, with 19 of them inhibiting the enzyme with apparent Ki values better than 50{micro}M. As our libraries continue to grow, a strategy of catching docking artifacts by rescoring with orthogonal methods may find wide use in the field. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/603966v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@c2f1a5org.highwire.dtl.DTLVardef@86bc58org.highwire.dtl.DTLVardef@1b739c7org.highwire.dtl.DTLVardef@325b93_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Small molecule FICD inhibitors suppress endogenous and pathologic FICD-mediated protein AMPylation

The AMP transferase, FICD, is an emerging drug target finetuning stress signaling in the endoplasmic reticulum (ER). FICD is a bi-functional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules to inhibit pathogenic FICD variants are lacking. Using an in-vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also efficiently inhibit pathogenic FICD variants and improve proinsulin processing in {beta} cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.

cell biology↗

bioRxiv2023

Guiding Discovery of Protein Sequence-Structure-Function Modeling

Protein engineering techniques are key in designing novel catalysts for a wide range of reactions. Although approaches vary in their exploration of the sequence-structure-function paradigm, they are often hampered by the labor-intensive steps of protein expression and screening. In this work, we describe the development and testing of a high throughput in silico sequence-structure-function pipeline using AlphaFold2 and Fast Fourier Transform docking that is benchmarked with enantioselectivity and reactivity predictions for an ancestral sequence library of fungal flavin-dependent monooxygenases. The predicted enantioselectivities and reactivities correlate well with previously described screens of an experimentally available subset of these proteins and capture known changes in enantioselectivity across the phylogenetic tree representing ancestorial proteins from this family. With this pipeline established as our functional screen, we apply ensemble decision tree models and explainable AI techniques to build sequence function models and extract critical residues within the binding site and the second sphere residues around this site. We demonstrate that the top-identified key residues in the control of enantioselectivity and reactivity correspond to experimentally verified residues. The in silico sequence-to-function pipeline serves as an accelerated framework to inform protein engineering efforts.

biophysics↗

bioRxiv2021

Norstictic acid is a selective allosteric transcriptional regulator

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (~900 [A]2) and have little topology, and thus do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprised of a highly dynamic loop flanking one canonical binding surface and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

biochemistry↗

bioRxiv2021

TMPRSS2 inhibitor discovery facilitated through an in silico and biochemical screening platform

The COVID-19 pandemic has highlighted the need for new antiviral targets, as many of the currently approved drugs have proven ineffective against mitigating SARS-CoV-2 infections. The host transmembrane serine protease TMPRSS2 is a highly promising antiviral target, as it plays a direct role in priming the spike protein before viral entry occurs. Further, unlike other targets such as ACE2, TMPRSS2 has no known biological role. Here we utilize virtual screening to curate large libraries into a focused collection of potential inhibitors. Optimization of a recombinant expression and purification protocol for the TMPRSS2 peptidase domain facilitates subsequent biochemical screening and characterization of selected compounds from the curated collection in a kinetic assay. In doing so, we demonstrate that serine protease inhibitors camostat, nafamostat, and gabexate inhibit through a covalent mechanism. We further identify new non-covalent compounds as TMPRSS2 protease inhibitors, demonstrating the utility of a combined virtual and experimental screening campaign in rapid drug discovery efforts.

biochemistry↗

bioRxiv2018

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing

HighlightsO_LIsmFRET, biochemistry and simulations probe co-transcriptional riboswitch folding\nC_LIO_LINascent RNA folding is disfavored by the DNA template and aided by RNAP\nC_LIO_LITranscriptional pausing is stabilized by RNA pseudoknot and destabilized by ligand\nC_LI\n\nSUMMARYNumerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to anti-bacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

biophysics↗