Biology subjects

Mittl, P. R. E.

Publications and source records attributed to Mittl, P. R. E..

2 recordsLinked to original sources

bioRxiv2026

Steric shielding of the KRAS4B hypervariable region enables isoform-specific inhibition of prenylation

Mutant KRAS is a potent oncogene, serving as a tumor driver in many solid human cancers. Current small-molecule inhibitors target the highly conserved G-domain, but to gain further mechanistic insight into the roles of different isoforms, we investigated the strategy of sterically shielding the unstructured hypervariable regions (HVRs). KRAS HVRs undergo a series of post-translational modifications that enable intracellular trafficking and membrane attachment. Previous attempts to drug KRAS by preventing its post-translational modification, based on inhibition of the involved prenylation enzymes have been largely unsuccessful. In this study, we explored the property of Designed Armadillo Repeat Proteins (dArmRPs) to specifically bind unstructured regions. We assembled a dArmRP to recognize the unstructured KRAS4B-HVR and developed it into a high-affinity binder by directed evolution. The resulting dArmRP recognizes the 14 C-terminal residues of unprocessed KRAS4B, thereby blocking the farnesyltransferase-binding epitope. This steric shielding disrupts KRAS4B post-translational modification and thereby significantly reduces its plasma membrane localization, while demonstrating complete selectivity over KRAS4A, NRAS, and HRAS. This work establishes the shielding of intrinsically disordered regions as a precise biochemical strategy to control protein function and provides an isoform-specific tool to dissect KRAS biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/712636v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1eddd7dorg.highwire.dtl.DTLVardef@1687a22org.highwire.dtl.DTLVardef@2a5407org.highwire.dtl.DTLVardef@f2d533_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical representation of how the unstructured KRAS4B-HVR is occupied by a dArmRP, making it inaccessible for the FTase.

biochemistry↗

bioRxiv2024

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage {varphi}2638A

AO_SCPLOWBSTRACTC_SCPLOWBacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage {varphi}2638A as a model to determine the interplay between its full-length endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via inter-amidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented impaired lytic activity, which could be partly restored through recombinant protein complementation of the missing isoform. Importantly, when applied as an antimicrobial protein against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. Drawing from our findings, we propose that SV isoform production provides its biological advantage upon endolysin entry to the periplasmic space to ensure optimal peptidoglycan degradation prior to cell wall lysis and progeny phage release.

molecular biology↗