Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition.

Proton-dependent oligopeptide transporters (POTs) are promiscuous transporters of the major facilitator superfamily that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several POT homologs have been studied extensively at the structural and molecular level. However, the molecular basis of recognition and transport of diverse peptide substrates has remained elusive. We present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe whether all 8400 natural di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides with compact hydrophobic residues are the best DtpB binders.

Crystal structure of the phospholipase A and acyltransferase 4 (PLAAT4) catalytic domain.

Phospholipase A and Acyltransferase 4 (PLAAT4) is a class II tumor suppressor, that also plays a role as a restrictor of intracellular Toxoplasma gondii infection through restriction of parasitic vacuole size. The catalytic N-terminal domain (NTD) interacts with the C-terminal domain (CTD), which is important for sub-cellular targeting and enzymatic function. The dynamics of the NTD main (L1) loop and the L2(B6) loop adjacent to the active site, have been shown to be important regulators of enzymatic activity. Here, we present the crystal structure of PLAAT4 NTD, determined from severely intergrown crystals using automated, laser-based crystal harvesting and data reduction technologies. The structure showed the L1 loop in two distinct conformations, highlighting a complex network of interactions likely influencing its conformational flexibility. Ensemble refinement of the crystal structure recapitulates the major correlated motions observed in solution by NMR. Our analysis offers useful insights on millisecond dynamics based on the crystal structure, complementing NMR studies which preclude structural information at this time scale.

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Three-Dimensional Structure of Full-Length NtrX, an Unusual Member of the NtrC Family of Response Regulators.

Bacteria sense and adapt to environmental changes using two-component systems. These signaling pathways are formed by a histidine kinase that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a two-component system formed by the histidine kinase NtrY and the RR NtrX that participates in sensing low oxygen tension and generating an adaptive response. NtrX is a modular protein with REC, AAA+, and DNA-binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article, we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs.

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization.

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS(239)S(240), we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno-blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.

A peptide derived from G0/G1 switch gene 2 acts as noncompetitive inhibitor of adipose triglyceride lipase.

The protein G0/G1 switch gene 2 (G0S2) is a small basic protein that functions as an endogenous inhibitor of adipose triglyceride lipase (ATGL), a key enzyme in intracellular lipolysis. In this study, we identified a short sequence covering residues Lys-20 to Ala-52 in G0S2 that is still fully capable of inhibiting mouse and human ATGL. We found that a synthetic peptide corresponding to this region inhibits ATGL in a noncompetitive manner in the nanomolar range. This peptide is highly selective for ATGL and does not inhibit other lipases, including hormone-sensitive lipase, monoacylglycerol lipase, lipoprotein lipase, and patatin domain-containing phospholipases 6 and 7. Because increased lipolysis is linked to the development of metabolic disorders, the inhibition of ATGL by G0S2-derived peptides may represent a novel therapeutic tool to modulate lipolysis.

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase.

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL's C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.

Adiponutrin functions as a nutritionally regulated lysophosphatidic acid acyltransferase.

Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.