Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltransferase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T . italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme’s lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

Staphylococcus aureus and other bacterial pathogens affix wall teichoic acids (WTAs) to their surface. These highly abundant anionic glycopolymers have critical functions in bacterial physiology and their susceptibility to β-lactam antibiotics. The membrane-associated TagA glycosyltranserase (GT) catalyzes the first-committed step in WTA biosynthesis and is a founding member of the WecB/TagA/CpsF GT family, more than 6,000 enzymes that synthesize a range of extracellular polysaccharides through a poorly understood mechanism. Crystal structures of TagA from T. italicus in its apo- and UDP-bound states reveal a novel GT fold, and coupled with biochemical and cellular data define the mechanism of catalysis. We propose that enzyme activity is regulated by interactions with the bilayer, which trigger a structural change that facilitates proper active site formation and recognition of the enzyme's lipid-linked substrate. These findings inform upon the molecular basis of WecB/TagA/CpsF activity and could guide the development of new anti-microbial drugs.

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The MAPK/ERK signaling pathway is one of the most heavily mutated in human cancers. In normal cells, the stimulation of receptor tyrosine kinases (e.g. EGFR) on the plasma membrane signals the GTP-loading and activation of RAS through a mechanism catalyzed by the guanine nucleotide exchange factor (GEF) Son of Sevenless 1 (SOS1). RAS activation, in turn, initiates the RAF-MEK-ERK kinase cascade which triggers many downstream signals that can affect gene transcription, protein translation, cell proliferation, and feedback regulation. Several factors regulate SOS1 GEF activity, including two autoinhibitory domains within its own multi-domain architecture. First, the N-terminal domains of SOS1 occlude the allosteric RAS-GTP binding site in the RAS exchanger motif—thereby precluding SOS1 GEF activity—and this inhibition is relieved upon membrane recruitment and PIP2 phospholipid binding. Second, the C-terminal Proline-Rich Region (PRR) of SOS1 also occludes allosteric RAS-GTP binding by an unknown mechanism and is relieved when the growth factor receptor-bound protein 2 (Grb2) binds to motifs in the PRR. While the independent functions of these domains and interactions to inhibition and activation of GEF activity of SOS1 are known, the molecular mechanisms are poorly understood. An additional negative feedback loop involves the phosphorylation of the SOS1 PRR at two sites by the ribosomal protein S6 kinase 1 (RSK1), a substrate of downstream ERK. These modifications create 14-3-3 binding sites on SOS1, where 14-3-3 binding downregulates GEF activity, possibly by obstructing Grb2 association with the SOS1 PRR and membrane localization of SOS1. This work reconstitutes the SOS1:Grb2 and the SOS1:14-3-3 protein complexes in vitro to study the molecular mechanisms underlying SOS1 activity regulation. Citation Format: Orlando E. Martinez, Jawahar Sudhamsu. Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A025.