Biology subjects

Sriramoju, M. K.

Publications and source records attributed to Sriramoju, M. K..

3 recordsLinked to original sources

bioRxiv2026

Substrate biasing in UCHL5 proteoforms

Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.

biochemistry↗

bioRxiv2024

A conformational fingerprint for amyloidogenic light chains.

Immunoglobulin light chain amyloidosis (AL) and multiple myeloma (MM) both share the overproduction of a clonal light chain (LC). However, while LCs in MM remain soluble in circulation, AL LCs misfold into toxic soluble species and amyloid fibrils that accumulate in organs, leading to distinct clinical manifestations. The significant sequence variability of LCs has hindered understanding of the mechanisms driving LC aggregation. Nevertheless, emerging biochemical properties, including dimer stability, conformational dynamics, and proteolysis susceptibility, distinguish AL LCs from those in MM under native conditions. This study aimed to identify a conformational fingerprint distinguishing AL from MM LCs. Using small-angle X-ray scattering (SAXS) under native conditions, we analyzed four AL and two MM LCs. We observed that AL LCs exhibited a slightly larger radius of gyration and greater deviations from X-ray crystallography-determined or predicted structures, reflecting enhanced conformational dynamics. SAXS data, integrated with molecular dynamics (MD) simulations, revealed a conformational ensemble where LCs adopt multiple states, with variable and constant domains either bent or straight. AL LCs displayed a distinct, low-populated, straight conformation (termed H state), which maximized solvent accessibility at the interface between constant and variable domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experimentally validated this H state. These findings reconcile diverse experimental observations and provide a precise structural target for future drug design efforts.

biophysics↗

bioRxiv2019

Identification of Escherichia coli ClpAP in regulating susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens

Type VI secretion system (T6SS) is an effector delivery system used by gram-negative bacteria to kill other bacteria or eukaryotic host to gain fitness. In Agrobacterium tumefaciens, T6SS has been shown to kill other bacteria such as Escherichia coli. Interestingly, the A. tumefaciens T6SS killing efficiency differs when using different E. coli strains as recipient cells. Thus, we hypothesize that a successful T6SS killing not only relies on attacker T6SS activity but also depends on recipient factors. A high-throughput interbacterial competition assay was employed to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. From the 3909 E. coli Keio mutants screened, 16 candidate mutants were filtered out. One strain, {Delta}clpP::Kan, showed ten times more resistant to T6SS-mediating killing but restored its susceptibility when complemented with clpP in trans. ClpP is a universal and highly conserved protease that exists in both prokaryotes and eukaryotic organelles. In E. coli, ClpP uses either ClpA or ClpX as an adaptor for substrate specificity. Therefore, the susceptibility of the {Delta}clpA::Kan and {Delta}clpX::Kan was also tested. The T6SS attack susceptibility of {Delta}clpA::Kan is at the same level as {Delta}clpP::Kan, while {Delta}clpX::Kan showed no difference as compared to that of wild-type E. coli BW25113. The data also suggest that ClpA-ClpP interaction, rather than its protease activity, is responsible for enhancing susceptibility to T6SS killing. This study highlights the importance of recipient factors in determining the outcome of T6SS killing.

microbiology↗