Andrew K. Lewis

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.

Physical Hydrogels Photo-Cross-Linked from Self-Assembled Macromers for Potential Use in Tissue Engineering

To engineer artificial extracellular matrices (ECMs) enabling degradation-independent cell migration that mimicked nonproteolytic cell migration through physically stabilized ECMs in biological systems, polymers having a hydrophilic chain flanked by a terminal self-assembling leucine zipper domain and a terminal photoreactive acrylate group were molecularly engineered to form photo-cross-linkable physical hydrogels. Physical association of the leucine zippers resulted in multifunctional macromers, which were photo-cross-linkable into hydrogels. Gel formation was confirmed by rheological measurements. The physical nature of the hydrogel networks was shown by hydrogel disassembly in denaturing solutions that disrupted the secondary structure of the leucine zippers. Outgrowth of encapsulated fibroblast aggregates was observed in these physical hydrogels but not observed in a control hydrogel in which leucine zippers were covalently linked. The collective properties of these hydrogels, including the physical nature, the photo-cross-linkable characteristic, and the flexibility for systematic engineering of material properties, will provide unique opportunities for tissue engineering.

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.

Modularly assembled porous cell-laden hydrogels

Porous cell-laden hydrogels have been modularly assembled to address the challenges in 3-dimensional tissue engineering. Microgels photolithographically fabricated from solutions of poly(ethylene glycol) diacrylate are assembled into large porous constructs in the presence of a polypeptide-based, physically bonded cross-linker. The assembly occurs through a physiologically permissive Michael-type addition reaction between the acrylate groups on the surface of the microgels and the thiol groups on the cross-linker. The constructs assembled from star-shaped microgels exhibit higher porosity, permeability, and pore interconnectivity than those formed from circle- and square-shaped microgels. The correlation between the properties of assembled constructs and the morphological features of microgels suggests the possibility for bottom-up modulation of the construct properties. The high pore interconnectivity revealed on the level of individual microgels suggests that these constructs are suitable for tissue engineering. Cells remain viable inside centimeter-sized constructs when cultured under perfusion. These constructs have the combined advantages of preformed porous scaffolds and in situ forming hydrogels in allowing enhanced mass transfer, uniform cell seeding, and protection of cells from excessive, non-physiological shear stress. Large constructs can be assembled in one step and have no limitations in size. This method will provide opportunities to create large 3-dimensional tissue engineered products.

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.

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer-sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers-undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor–receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

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.