Venkatasubramanian Dharmarajan

Conformational states of the full-length glucagon receptor.

Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide.

The mixed lineage leukemia protein-1 (MLL1) catalyzes histone H3 lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (retinoblastoma-binding protein-5), and the Ash2L (absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the “Win” or WDR5 interaction motif, that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-Å crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that Arg-3765 of MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes Arg-3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.

A Conserved Arginine-containing Motif Crucial for the Assembly and Enzymatic Activity of the Mixed Lineage Leukemia Protein-1 Core Complex

The mixed lineage leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD repeat protein-5 (WDR5), retinoblastoma-binding protein-5 (RbBP5), and the absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins, we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine-containing motif, called the “Win” or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5s recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.

Estrogen receptor alpha somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating function-2 binding conformation

Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition.

Structure of the full-length glucagon class B G-protein-coupled receptor.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen–deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1 ) core complex

Abstract Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.

HDX-MS guided drug discovery: small molecules and biopharmaceuticals.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS or DXMS) has emerged as an important tool for the development of small molecule therapeutics and biopharmaceuticals. Central to these advances have been improvements to automated HDX-MS platforms and software that allow for the rapid acquisition and processing of experimental data. Correlating the HDX-MS profile of large numbers of ligands with their functional outputs has enabled the development of structure activity relationships (SAR) and delineation of ligand classes based on functional selectivity. HDX-MS has also been applied to address many of the unique challenges posed by the continued emergence of biopharmaceuticals. Here we review the latest applications of HDX-MS to drug discovery, recent advances in technology and software, and provide perspective on future outlook.

Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a significant clinical problem for estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Inhibiting AMPylation: A novel screen to identify the first small molecule inhibitors of protein AMPylation

Enzymatic transfer of the AMP portion of ATP to substrate proteins has recently been described as an essential mechanism of bacterial infection for several pathogens. The first AMPylator to be discovered, VopS from Vibrio parahemolyticus, catalyzes the transfer of AMP onto the host GTPases Cdc42 and Rac1. Modification of these proteins disrupts downstream signaling events, contributing to cell rounding and apoptosis, and recent studies have suggested that blocking AMPylation may be an effective route to stop infection. To date, however, no small molecule inhibitors have been discovered for any of the AMPylators. Therefore, we developed a fluorescence-polarization-based high-throughput screening assay and used it to discover the first inhibitors of protein AMPylation. Herein we report the discovery of the first small molecule VopS inhibitors (e.g., calmidazolium, GW7647, and MK886) with Kis ranging from 6 to 50 μM and upward of 30-fold selectivity versus HYPE, the only known human AMPylator.