Biology subjects

Saran, A.

Publications and source records attributed to Saran, A..

4 recordsLinked to original sources

bioRxiv2026

Structural and mechanistic basis of ubiquitous bacterial kinase signaling identifies PorX as a noncanonical substrate in Porphyromonas gingivalis

Protein phosphorylation enables bacteria to coordinate regulatory networks underlying virulence and environmental adaptation. The ubiquitous bacterial kinase family comprises atypical kinases with dual serine threonine and tyrosine specificity, yet their structural organization, catalytic mechanisms, and physiological roles remain incompletely defined. In the anaerobic oral pathogen Porphyromonas gingivalis, the sole UbK homolog, UbK1, was previously shown to phosphorylate the orphan response regulator RprY, linking UbK1 to virulence-associated pathways. Here, we present the crystal structure of UbK1, revealing the conserved Walker A, HxDxYR, SPT/S, and EW motifs arranged around the ATP-binding site. Structure-guided mutagenesis establishes essential roles for these motifs in ATP hydrolysis and kinase activity. Phosphosite mapping identifies multiple autophosphorylation sites, with the flexible SPT/S loop showing the strongest occupancy and supporting a model in which loop centered autophosphorylation is a major feature of UbK1 cycling while additional sites arise through intermolecular phosphotransfer in trans. Consistent with this, biochemical assays demonstrate that UbK1 undergoes autophosphorylation both in cis and in trans, arguing against a strictly intramolecular autokinase mechanism. Using conserved gene neighborhood analysis, we identified the orphan response regulator PorX as a previously unrecognized UbK1 substrate, consistent with the relaxed substrate specificity reported for UbK homologs. UbK1 catalyzes PorX transphosphorylation in vitro at a single tyrosine residue within the receiver domain, independent of PorX oligomeric state. In vivo, ubk1 deletion causes a modest reduction in gingipain secretion, whereas mutation of the UbK1 dependent PorX phosphosite does not measurably affect T9SS mediated cargo export, indicating that UbK1-PorX signaling is separable from PorX canonical gingipain secretion output. Together, these findings establish a structural and mechanistic framework for UbK1 function, expand the UbK substrate repertoire, and support a model in which UbK1 contributes to regulatory pathways in P. gingivalis that extend beyond canonical secretion associated outputs.

molecular biology↗

bioRxiv2025

Cyclo-stationary distributions of mRNA and Protein counts for random cell division times

There is a long history of using experimental and computational approaches to study noise in single-cell levels of mRNA and proteins. The noise originates from a myriad of factors: intrinsic processes of gene expression, partitioning errors during division, and extrinsic effects, such as, random cell-cycle times. Although theoretical methods are well developed to analytically understand full statistics of copy numbers for fixed or Erlang distributed cell cycle times, the general problem of random division times is still open. For any random (but uncorrelated) division time distribution, we present a method to address this challenging problem and obtain exact series representations of the copy number distributions in the cyclo-stationary state. We provide explicit cell age-specific and age-averaged results, and analyze the relative contribution to noise from intrinsic and extrinsic sources. Our analytical approach will aid the analysis of single-cell expression data and help in disentangling the impact of variability in division times.

systems biology↗

bioRxiv2024

Unveiling the Molecular Mechanisms of the Type-IX Secretion System's Response Regulator: Structural and Functional Insights

The Type-IX secretion system (T9SS) is a nanomachinery utilized by bacterial pathogens to facilitate infection. The system is regulated by a signaling cascade serving as its activation switch. A pivotal member in this cascade, the response regulator protein PorX, represents a promising drug target to prevent the secretion of virulence factors. Here, we provide a comprehensive characterization of PorX both in vitro and in vivo. First, our structural studies revealed PorX harbours a unique enzymatic effector domain, which, surprisingly, shares structural similarities with the alkaline phosphatase superfamily, involved in nucleotide and lipid signaling pathways. Importantly, such pathways have not been associated with the T9SS until now. Enzymatic characterization of PorXs effector domain revealed a zinc-dependent phosphodiesterase activity, with active site dimensions suitable to accommodate a large substrate. Unlike typical response regulators that dimerize via their receiver domain upon phosphorylation, we found that zinc can also induce conformational changes and promote PorXs dimerization via an unexpected interface. These findings suggest that PorX can serve as a cellular zinc sensor, broadening our understanding of its regulatory mechanisms. Despite the strict conservation of PorX in T9SS-utilizing bacteria, we demonstrate that PorX is essential for virulence factors secretion in Porphyromonas gingivalis and affects metabolic enzymes secretion in the non-pathogenic Flavobacterium johnsoniae, but not for the secretion of gliding adhesins. Overall, this study advances our structural and functional understanding of PorX, highlighting its potential as a druggable target for intervention strategies aimed at disrupting the T9SS and mitigating virulence in pathogenic species.

biochemistry↗

bioRxiv2021

Structural basis for feedforward control in the PINK1/parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinsons disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

biochemistry↗