Christopher C. Valley

The Methionine-aromatic Motif Plays a Unique Role in Stabilizing Protein Structure

Background: The interaction between methionine and aromatic residues in protein complexes is poorly understood. Results: The Met-aromatic motif is prevalent in known protein structures and stabilizes TNF ligand-receptor binding interactions. Conclusion: The Met sulfur-aromatic binding motif provides additional stabilization over purely hydrophobic interactions and at longer distances. Significance: This motif is prevalent and may be associated with a number of mutation- and age-associated diseases. Of the 20 amino acids, the precise function of methionine (Met) remains among the least well understood. To establish a determining characteristic of methionine that fundamentally differentiates it from purely hydrophobic residues, we have used in vitro cellular experiments, molecular simulations, quantum calculations, and a bioinformatics screen of the Protein Data Bank. We show that approximately one-third of all known protein structures contain an energetically stabilizing Met-aromatic motif and, remarkably, that greater than 10,000 structures contain this motif more than 10 times. Critically, we show that as compared with a purely hydrophobic interaction, the Met-aromatic motif yields an additional stabilization of 1–1.5 kcal/mol. To highlight its importance and to dissect the energetic underpinnings of this motif, we have studied two clinically relevant TNF ligand-receptor complexes, namely TRAIL-DR5 and LTα-TNFR1. In both cases, we show that the motif is necessary for high affinity ligand binding as well as function. Additionally, we highlight previously overlooked instances of the motif in several disease-related Met mutations. Our results strongly suggest that the Met-aromatic motif should be exploited in the rational design of therapeutics targeting a range of proteins.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Induces Death Receptor 5 Networks That Are Highly Organized

Background: Whether ligand-induced clusters of DR5 have a specific structural organization is unknown. Results: Ligand binding results in the formation of death receptor dimers that exist within high molecular weight networks. Conclusion: Ligand-induced DR5 clusters are highly organized networks formed through dimerization of receptor trimers. Significance: The biophysical character of DR5 networks may have implications for future rational design of DR5-targeted therapeutics. Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

HuR’s post-transcriptional regulation of death receptor 5 in pancreatic cancer cells

Apoptosis is one of the core signaling pathways disrupted in pancreatic ductal adenocarcinoma (PDA). Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF)-receptor superfamily that is expressed in cancer cells. Binding of TNF-related apoptosis-inducing ligand (TRAIL) to DR5 is a potent trigger of the extrinsic apoptotic pathway, and numerous clinical trials are based on DR5-targeted therapies for cancer, including PDA. Human antigen R (HuR), an RNA-binding protein, regulates a select number of transcripts under stress conditions. Here we report that HuR translocates from the nucleus to the cytoplasm of PDA cells upon treatment with a DR5 agonist. High doses of DR5 agonist induce cleavage of both HuR and caspase 8. HuR binds to DR5 mRNA at the 5′-untranslated region (UTR) in PDA cells in response to different cancer-associated stressors and subsequently represses DR5 protein expression; silencing HuR augments DR5 protein production by enabling its translation and thus enhances apoptosis. In PDA specimens (n = 53), negative HuR cytoplasmic expression correlated with elevated DR5 expression (odds ratio 16.1, p < 0.0001). Together, these data demonstrate a feedback mechanism elicited by HuR-mediated repression of the key apoptotic membrane protein DR5.

TNFR1 signaling is associated with backbone conformational changes of receptor dimers consistent with overactivation in the R92Q TRAPS mutant

The widely accepted model for tumor necrosis factor 1 (TNFR1) signaling is that ligand binding causes receptor trimerization, which triggers a reorganization of cytosolic domains and thus initiates intracellular signaling. This model of stoichiometrically driven receptor activation does not account for the occurrence of ligand independent signaling in overexpressed systems, nor does it explain the constitutive activity of the R92Q mutant associated with TRAPS. More recently, ligand binding has been shown to result in the formation of high molecular weight, oligomeric networks. Although the dimer, shown to be the preligand structure, is thought to remain present within ligand-receptor networks, it is unknown whether network formation or ligand-induced structural change to the dimer itself is the trigger for TNFR1 signaling. In the present study, we investigate the available crystal structures of TNFR1 to explore backbone dynamics and infer conformational transitions associated with ligand binding. Using normal-mode analysis, we characterize the dynamic coupling between the TNFR1 ligand binding and membrane proximal domains and suggest a mechanism for ligand-induced activation. Furthermore, our data are supported experimentally by FRET showing that the constitutively active R92Q mutant adopts an altered conformation compared to wild-type. Collectively, our results suggest that the signaling competent architecture is the receptor dimer and that ligand binding modifies domain mobilities intrinsic to the receptor structure, allowing it to sample a separate, active conformation mediated by network formation.

Oxidation increases the strength of the methionine-aromatic interaction

Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. Using quantum mechanical calculations, we show that oxidation increases the strength of the methionine-aromatic interaction motif—a driving force for protein folding and protein-protein interaction—by 0.5 – 1.4 kcal/mol. We find that non-hydrogen bonded interactions between dimethyl sulfoxide (a methionine analog) and aromatic groups are enriched in both the Protein Data Bank and Cambridge Structural Database. Thermal denaturation and NMR experiments on model peptides demonstrate that oxidation of methionine stabilizes the interaction by 0.5–0.6 kcal/mol. We confirm the biological relevance of these findings through a combination of cell biology, electron paramagnetic resonance spectroscopy and molecular dynamics simulations on 1) calmodulin structure and dynamics and 2) lymphotoxin-α/TNFR1 binding. Thus, the methionine-aromatic motif is a determinant of protein structural and functional sensitivity to oxidative stress.

Piecing it together: Unraveling the elusive structure-function relationship in single-pass membrane receptors

The challenge of crystallizing single-pass plasma membrane receptors has remained an obstacle to understanding the structural mechanisms that connect extracellular ligand binding to cytosolic activation. For example, the complex interplay between receptor oligomerization and conformational dynamics has been, historically, only inferred from static structures of isolated receptor domains. A fundamental challenge in the field of membrane receptor biology, then, has been to integrate experimentally observable dynamics of full-length receptors (e.g. diffusion and conformational flexibility) into static structural models of the disparate domains. In certain receptor families, e.g. the ErbB receptors, structures have led somewhat linearly to a putative model of activation. In other families, e.g. the tumor necrosis factor (TNF) receptors, structures have produced divergent hypothetical mechanisms of activation and transduction. Here, we discuss in detail these and other related receptors, with the goal of illuminating the current challenges and opportunities in building comprehensive models of single-pass receptor activation. The deepening understanding of these receptors has recently been accelerated by new experimental and computational tools that offer orthogonal perspectives on both structure and dynamics. As such, this review aims to contextualize those technological developments as we highlight the elegant and complex conformational communication between receptor domains. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function.

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix-helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Abstract 4100: HuR regulates death receptor-5 (DR5, TRAIL-R2)-targeted treatment of pancreatic cancer cells

Apoptosis has been identified as a core signaling pathway disrupted in pancreatic ductal adenocarcinoma (PDA) tumorigenesis. Death Receptor 5 (DR5, TRAIL-R2) is a membrane bound protein that initiates the extrinsic apoptotic pathway upon ligand exposure and is currently being explored as a ‘druggable’ target in multiple cancers including PDA. Identifying a mechanism that regulates DR5 in the tumor microenvironment (e.g. hypoxia, chemotherapeutic exposure) is critical for optimizing DR5 based-therapies. Human antigen R (HuR), an RNA binding protein, controls post-transcriptional gene expression by binding to specific regions of 3’and 5’ UTRs of mRNA target genes. Previously, HuR, a pro-survival molecule, has been shown to play an important role in the intrinsic apoptotic pathway. We identified DR5 mRNA as a HuR target in PDA cells and explored the significance of HuR9s role in functionally regulating the extrinsic apoptotic pathway in PDA cells. We also explored HuR as a modulator of DR5-targeted therapy for the treatment of PDA. Ribonucleoprotein immunoprecipitation (RNP-IP) assays were performed on PDA cells using HuR antibody (Ab) compared to a control (IgG Ab) under stress conditions, 3 hours with 1μM of the standard of care drug for PDA, gemcitabine; and 75 μM of a PARP inhibitor (PARPi). mRNA was converted to cDNA using RT-PCR, and then analyzed by qPCR. DR5 mRNA was validated as a HuR target with a 6-fold greater binding to HuR compared to the control. Strikingly, this binding increases 12- and 24-fold upon treatment with gemcitabine and the PARPi respectively. Silencing HuR expression, through siRNA transfections, leads to an increase of DR5 protein expression at 24 and 48 hours in multiple PDA cell lines. Additionally, silencing of HuR significantly enhances the action of a DR5-specific monoclonal Ab (0.8 mg/mL) against PDA cells within 36 hours (a 20% detected increase in cell death compared to control cells), most likely due to an enhanced availability of the DR5 receptor. Finally, in a training set of PDA clinical specimens, we found a significant inverse correlation between high/low HuR cytoplasmic expression and low/high DR5 levels (p value=0.03). In over 80% (26 of 31) of the specimens HuR cytoplasmic levels inversely correlated with DR5 expression levels, providing further evidence that elevated cytoplasmic HuR is repressing DR5 protein levels in patient tumor cells. In sum, we have shown that ‘activated HuR’ represses DR5 protein expression in PDA cells. Therefore, we conclude that low cytoplasmic HuR levels allow for greater availability of the target DR5, and will thus accordingly enhance the efficacy of DR5-targeted therapy. Thus, manipulating and/or utilizing HuR expression levels may serve as a clinically informative tool for optimizing DR5-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4100. doi:10.1158/1538-7445.AM2011-4100