Protein interactions between MAP kinases and substrates, activators, and scaffolding proteins are regulated by docking site motifs, one containing basic residues proximal to Leu-X-Leu (DEJL) and a second containing Phe-X-Phe (DEF). Hydrogen exchange mass spectrometry was used to identify regions in MAP kinases protected from solvent by docking motif interactions. Protection by DEJL peptide binding was observed in loops spanning beta7-beta8 and alphaD-alphaE in p38alpha and ERK2. In contrast, protection by DEF binding to ERK2 revealed a distinct hydrophobic pocket for Phe-X-Phe binding formed between the P+1 site, alphaF helix, and the MAP kinase insert. In inactive ERK2, this pocket is occluded by intramolecular interactions with residues in the activation lip. In vitro assays confirm the dependence of Elk1 and nucleoporin binding on ERK2 phosphorylation, and provide a structural basis for preferential involvement of active ERK in substrate binding and nuclear pore protein interactions.

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.

WNK kinases comprise a small group of unique serine/threonine protein kinases that have been genetically linked to pseudohypoaldosteronism type II, an autosomal dominant form of hypertension. Here we present the structure of the kinase domain of WNK1 at 1.8 A resolution, solved in a low activity conformation. A lysine residue (Lys-233) is found in the active site emanating from strand beta2 rather than strand beta3 as in other protein kinases. The activation loop adopts a unique well-folded inactive conformation. The conformations of the P+1 specificity pocket, the placement of the conserved active site threonine (Thr-386), and the exterior placement of helix C, contribute to the low activity state. By homology modeling, we identified two hydrophobic residues in the substrate-binding groove that contribute to substrate specificity. The structure of the WNK1 catalytic domain, with its unique active site, may help in the design of therapeutic reagents for the treatment of hypertension.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with and characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased and stability of TREM2 contribute to loss of function in disease.

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast twohybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a Cterminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.WNKs (With No lysine (K)) are serine/threonine protein kinases implicated in regulating ion permeability in epithelia (1-4). There are four mammalian WNK family members (5, 6), and mutations in two of them, WNK1 and WNK4, have been linked to a hereditary form of human hypertension known as pseudohypoaldosteronism type II (1). This discovery has provoked a broad search for physiological mechanisms by which WNKs regulate blood pressure, as well as relationships between mutations in WNKs and other types of hypertension (7,8). Mutations in WNK4 are in the coding sequence, whereas mutations in WNK1 are intronic and cause overexpression of the wild-type protein (1). Because we originally isolated cDNAs encoding WNK1 (5), we have been working to understand signal transduction events mediated by WNK1 and the biochemical mechanisms utilized by it that might modulate membrane permeability (9, 10).WNKs are unique because the lysine required for phosphoryl transfer lies in the phosphate anchor ribbon (kinase subdomain I) instead of ␤ strand 3 (kinase subdomain II), its position in all other members of the protein kinase superfamily (5). The kinase domains of WNKs are located near their N termini. WNKs contain a conserved autoinhibitory domain, first identified in WNK1, and two predicted coiled-coil domains, which are located C-terminal to the kinase domain (6, 9, 11). The WNK1 autoinhibitory domain (residues 485-555) reduces WNK1 autophosphorylation and substrate phosphorylation (9). Similarly, the WNK4 autoinhibitory domain (residues 444 -518) was reported to inhibit WNK1 autophosphorylation, suggesting WNKs may modulate the kinase activity of other family members (11).In this study, we have continued to characterize the biochemical properties and regulation of WNK1 that may ...