Biology subjects

Ramu, M.

Publications and source records attributed to Ramu, M..

4 recordsLinked to original sources

bioRxiv2026

Functional coupling between the a4 - a5 loop and allosteric site on MKP5 is a critical determinant of catalysis

Dual-specificity phosphatases (DUSPs) that inactivate mitogen-activated protein kinases (MAPKs) are called MAPK phosphatases (MKPs). The stress-responsive MKP, MKP5, dephosphorylates p38 MAPK and c-Jun NH2-terminal kinase (JNK). Previously, we identified an allosteric compound that binds to the site within the phosphatase domain of MKP5 that is critical for the binding and dephosphorylation of both p38 MAPK and JNK. The allosteric site, comprised of the 4-5 loop, is an essential region for transmitting MAPK binding to the catalytic site. Here, we examine the contribution of additional structural rearrangements that occur upon allosteric site engagement. We show that binding of an inhibitor that engages the MKP5 allosteric site induces conformational changes in the 4-5 loop. Mutants of residues in this loop inhibited enzymatic activity, and some mutants exhibited changes in dynamics, indicating that this loop has a significant structural and dynamic role in controlling MKP5 catalysis. Enzymatic and NMR studies supported the interpretation that conformational changes and dynamics in the 4-5 loop are required for enzymatic function. Additionally, alanine mutants of R442 (4-5 loop), the catalytic base D377 ({beta}4-2 loop), and Q409A and S413A ({beta}5-3 loop) disrupted catalytic activity. These results highlight the 4-5, {beta}4-2, and {beta}5-3 loops as structurally, dynamically, and functionally interconnected for communication between the allosteric and enzymatic sites.

biochemistry↗

bioRxiv2024

Deciphering the molecular mechanism associated with the interaction of TULP3 and Histone deacetylases, SIRT1 and SIRT2

Tubby-like protein-3 (TULP3), a member of the tubby family, is recognized for its role in ciliary trafficking, linking membrane-associated proteins to the intraflagellar transport complex A (IFT-A). Mammalian tubby family proteins (TULP1-4), featuring a C-terminal tubby domain, play crucial roles in intracellular transport, signaling, cell differentiation, and motility. TULP3 is also associated with renal and hepatic fibrocystic disease and liver, kidney, and heart degeneration. However, the modulation of TULP3 function and its interacting partners in the cell still need to be understood. Recent studies have indicated interactions between TULP3 and various proteins, including acetylating and deacetylating enzymes. In this study, we investigate the interaction of TULP3 with Sirtuins, SIRT1, and SIRT2 using biochemical and biophysical techniques. Our findings revealed that both the N- and C-terminal domains of TULP3 are necessary for the interaction with SIRT1 and SIRT2. Furthermore, we have identified that TULP3 is not a deacetylation substrate for SIRT1.

biochemistry↗

bioRxiv2024

Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase

Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.

biochemistry↗

bioRxiv2023

Structures of Arabidopsis thaliana MDL Proteins and Synergistic Effects with the Cytokine MIF on Human Receptors

Vertebrates have developed effective immune mechanisms to fight microbial attacks, relying on a sophisticated network of innate and adaptive responses, a circulatory system, and numerous orchestrating soluble mediators such as cytokines. Mammalian macrophage migration inhibitory factor (MIF) and its paralog D-dopachrome tautomerase (D-DT/MIF-2) are multifunctional inflammatory cytokines with chemokine-like properties that modulate immunity. Plants possess orthologous MIF/D-DT-like (MDL) proteins, whose function is largely unexplored. Driven by the previous discovery of cross-kingdom mimicry of plant (Arabidopsis thaliana) MDL proteins and human MIF receptor signaling, we here characterized the structures of the three A. thaliana MDLs by X-ray crystallography and explored the mechanism underlying the interplay between plant MDLs, human MIF, and its receptors. We obtained high-resolution structures at 1.56 [A], 1.40 [A], and 2.00 [A] resolution for MDL1, MDL2, and MDL3, respectively, revealing a typical trimeric assembly and a high three-dimensional similarity to human MIF. Although residues at the catalytic site of the three MDLs show high identity to human MIF, the proteins showed low tautomerase activity for the substrate 4-hydroxyphenylpyruvate (HPP). Structural differences likely explain the enzymatic inactivity of plant MDLs for HPP. Strikingly, employing in vitro, in vivo, and in planta test systems, we found that MIF and MDL proteins interact with each other and have the capacity to form hetero-oligomeric complexes. The functional consequences of this interaction were demonstrated applying a yeast-based reporter system specific for the MIF chemokine receptors CXCR2 and CXCR4. MDLs not only triggered receptor signaling on their own, but exhibited pronounced synergism regarding the activation of the CXCR2- and CXCR4-dependent signaling pathways, when co-applied with MIF. These findings were substantiated by the co-administration of pharmacological inhibitors that either disrupt MIF receptor binding or block the catalytic cavity. Moreover, biochemical and biophysical experiments using an allosteric oligomer-specific MIF inhibitor established hexa-oligomer formation between MIF and MDLs as the putative basis for the synergistic effect. Our results are the starting point for a mechanistic understanding of the immunomodulatory activity of a family of highly conserved plant proteins. One Sentence SummaryA. thaliana MDLs and human macrophage inhibitory factor interact with each other and induce synergistic effects in activating CXCR2 and CXCR4.

immunology↗