Biology subjects

Sreenivasan, S.

Publications and source records attributed to Sreenivasan, S..

3 recordsLinked to original sources

bioRxiv2023

Fructose-1-kinase has pleiotropic roles in Escherichia coli

In Escherichia coli, the master transcription regulator Catabolite Repressor Activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a {Delta}fruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. The {Delta}fruK strain also alters biofilm formation. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other {gamma}-proteobacteria, which conserve both Cra and FruK.

microbiology↗

bioRxiv2023

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new rule?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies - which would also detect tuneability - used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: The ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. Here, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITAs AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITAs function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behaviour for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

biochemistry↗

bioRxiv2022

Identification of a covert evolutionary pathway between two protein folds

Although homologous protein sequences are expected to adopt similar structures, some amino acid substitutions can interconvert -helices and {beta}-sheets. Such fold switching may have occurred over evolutionary history, but supporting evidence has been limited by the: (1) abundance and diversity of sequenced genes, (2) quantity of experimentally determined protein structures, and (3) assumptions underlying the statistical methods used to infer homology. Here, we overcame these barriers by applying multiple statistical methods to a family of ~600,000 bacterial response regulator proteins. We found that their homologous DNA-binding subunits assume divergent structures: helix-turn-helix versus -helix+{beta}-sheet (winged helix). Phylogenetic analyses, ancestral sequence reconstruction, and AlphaFold2 models indicated that amino acid substitutions facilitated a switch from helix-turn-helix into winged helix. This structural transformation likely expanded DNA-binding specificity. Our approach uncovers an evolutionary pathway between two protein folds and provides methodology to identify secondary structure switching in other protein families.

bioinformatics↗