Biology subjects

McShan, A. C.

Publications and source records attributed to McShan, A. C..

6 recordsLinked to original sources

bioRxiv2026

Tertiary structure of a Nif11-like leader peptide mediates protein-protein interactions with a RiPP biosynthetic enzyme

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced from precursor peptides that typically contain a short, unstructured N-terminal leader region that engages RiPP biosynthetic enzymes through peptide-protein contacts. A distinct subclass of leader peptides, the nitrogen-fixing protein 11-like leader peptides (Nif11-LPs), are unusually long and conspicuously enriched in cyanobacteria. No atomic structures of Nif11-LPs have been reported. Here, we report the solution structure of MppEleader, a Nif11-LP from the marine cyanobacterium Moorena producens PAL. MppEleader adopts a bipartite architecture with a rigid N-terminal tetrahelical bundle stabilized around a hydrophobic nucleus, followed by a dynamic, largely disordered C-terminal region containing a conserved L(X)L motif. We show that the -ketoglutarate/non-heme iron enzyme MppG dihydroxylates the macrocyclized MppEcore peptide in a strictly leader-dependent manner. NMR titrations and structure-guided mutagenesis demonstrate that both the tetrahelical hydrophobic nucleus and the L(X)L motif of MppE make direct, functionally required contacts with MppG, including its RiPP recognition element (RRE) domain. An AlphaFold3-guided structural model rationalizes these contacts. Together, these findings establish a tertiary structure-dependent protein-protein interaction paradigm for RiPP core peptide modification, distinguishing Nif11-LP recognition from canonical short-leader mechanisms and expanding the understanding of substrate recognition in RiPP biosynthesis.

biochemistry↗

bioRxiv2026

SILCS-Guided Feature Encoding Expands Ligand Recognition at an HBV Core Protein Interface

Hepatitis B virus (HBV) infection depends on coordinated capsid assembly and virion production. A hydrophobic pocket at the intradimer interface of the HBV core protein has been linked to secretion phenotypes and shown to bind small molecules, but its interaction landscape remains poorly defined. Here, we combine atomistic molecular dynamics (MD) simulations, fragment mapping, pharmacophore modeling, and biophysical binding assays to characterize this pocket and identify ligands with binding modes that extend beyond the known pocket. This workflow narrowed an initial library of approximately 4.7 million compounds to eight candidates for experimental evaluation by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). Of these eight, compound B3 showed detectable STD NMR signals, concentration-dependent SPR binding, and an MD-supported binding mode that retained hydrophobic-pocket anchoring while also extending toward the spike-proximal loop. Together, these results illustrate how dynamic fragment mapping can identify ligand candidates that engage broader interaction landscapes at protein-interface pockets.

biophysics↗

bioRxiv2026

Potent substoichiometric inhibition of alpha-synuclein aggregation by de novo oligomer-binding proteins

The intrinsically disordered, non-amyloid-{beta} component domain of -synuclein (Syn) drives aggregation in Parkinson's disease but lacks a stable epitope for structure-based drug design. We used deep learning-based protein design to generate de novo binders capturing this region in a {beta}-strand conformation. Three designs bound Syn with nanomolar affinities, with solution NMR spectroscopy supporting the intended fold. All three suppressed fibril formation at substoichiometric ratios, and fitting of aggregation kinetics resolved the microscopic steps inhibited by each. Notably, one binder preferentially engaged oligomers and inhibited secondary nucleation, both closely linked to toxicity. In cells, all three suppressed seeded Syn assembly, with the oligomer binder being the most effective. Together, these findings establish a set of molecules that target distinct aggregation intermediates and mechanistically reshape Syn assembly.

biophysics↗

bioRxiv2020

TAPBPR Promotes Antigen Loading on MHC-I Molecules Using a Peptide Trap

Chaperones tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 residue loop in TAPBPR (corresponding to 11 residues in tapasin), which has been proposed to actively compete with incoming peptides by forming direct contacts in the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for the chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward peptide editing are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner. Detailed biophysical characterization by ITC, FP and NMR reveals that the loop does not interact with the peptide-deficient MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic trap. Our results suggest that the longer loop of TAPBPR lowers the affinity threshold for peptide selection, to promote loading within subcellular compartments of reduced peptide concentration and to prevent disassembly of high affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

immunology↗

bioRxiv2019

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAPBPR associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized towards one side of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the 2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at \"hotspot\" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the 1/2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of i) chaperoning unstable MHC-I molecules at early stages of their folding process, akin to a holdase with broad allele-specificity, and ii) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, towards editing Sithe repertoire of displayed antigens.\n\nSignificance StatementThe human population contains thousands of MHC-I alleles, showing a range of dependencies on molecular chaperones for loading of their peptide cargo, which are then displayed on the cell surface for T cell surveillance. Using the chaperone TAPBPR as a model, we combine deep mutagenesis with functional and biophysical data, especially solution NMR, to provide a complete view of the molecular determinants of chaperone recognition. Our data provide significant evidence that localized protein motions define the intrinsic ability of MHC-I molecules to interact with chaperones. The importance of MHC-I dynamics unifies all our findings, with broad recognition of conformationally unstable, nascent MHC-I molecules becoming restricted to a smaller set of MHC-I alleles that retain relevant dynamic motions in their folded state.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC=\"FIGDIR/small/779777v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@19da2fforg.highwire.dtl.DTLVardef@383607org.highwire.dtl.DTLVardef@63171eorg.highwire.dtl.DTLVardef@182bf14_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone\nC_LIO_LIAnalysis of s-ms timescale conformational dynamics by methyl NMR reveals allele-specific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules\nC_LIO_LIs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, \"excited state\" conformation\nC_LIO_LIRestriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane\nC_LI

immunology↗

bioRxiv2019

De novo design of proteins with two isoenergetic but structurally divergent ground states

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used to de novo design proteins that fold to a single state with a deep free energy minima (Huang et al., 2016), and to reengineer natural proteins to alter their dynamics (Davey et al., 2017) or fold (Alexander et al., 2009), the de novo design of closely related sequences which adopt well-defined, but structurally divergent structures remains an outstanding challenge. Here, we design closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations -- one short ([~]66 [A] height) and the other long ([~]100 [A] height) -- reminiscent of the conformational transition of viral fusion proteins (Ivanovic et al., 2013; Podbilewicz, 2014; Skehel and Wiley, 2000). Crystallographic and NMR spectroscopic characterization show that both the short and long state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large scale conformational switches between structurally divergent forms.

biochemistry↗