My laboratory is investigating structural and functional aspects of cell-surface receptor recognition and activation, in receptor/ligand systems relevant to human health and disease. The general question we are endeavoring to address is how the extracellular engagement of a ligand by a receptor is structurally coupled to the activation of intracellular signaling cascades. The molecular mechanisms by which information is communicated from the outside to the inside of a cell, by means of a membrane-bound receptor,  are not well understood.  Yet, cell-surface receptor activation is fundamental to essentially every vital physiological function.  On one hand, we are making progress in understanding molecular recognition between proteins through biophysical studies of protein-protein complexes.  However, the molecular bases for the subsequent transduction of an activation signal, upon receptor engagement, remains largely speculative for even the most intensively studied receptor systems.  Models to explain the generation of an activation signal generally invoke either ligand-induced receptor clustering, conformational change, or a combination of both.  These models have been derived from structural analysis of receptor extracellular and intracellular domains which have been reconciled with functional data.  Eventually, clear answers to this question will require structural studies of full-length receptors, including membrane-spanning domains, both in the presence and absence of ligand.   Our goal is to understand the structural link between ligand recognition and receptor activation for a variety of receptor systems.  To gain insight into this process we are determining crystal structures of receptors in complex with their ligands, in parallel with complementary biochemical experiments.

Receptor structural biology necessarily requires both structural and functional studies, and so the experimental strategy in my laboratory is to produce reagents which we can utilize for both approaches.  This strategy is manifested in an interplay between protein engineering and x-ray crystallography.  The production, or expression of recombinant receptors and their subsequent crystallization is the major technical challenge we face in the laboratory.  Hence, a significant effort is dedicated to designing biologically active, crystallizable proteins and subsequently expressing sufficient quantities of these proteins for our experiments.  The proper engineering of these proteins usually requires significant insight into their functional properties such as oligomerization states, interactions with accessory proteins, glycosylation, membrane associations, and ligand binding affinities. Elucidating these functional properties is an integral component of the project, and critical to designing appropriate crystallization and expression strategies for the macromolecular assemblies we study. Upon successful expression of the desired receptor/ligand complexes, we apply methods such as isothermal titration calorimetry to measure binding thermodynamics, surface plasmon resonance to measure binding kinetics, and a variety of other biophysical methods aimed at probing the details of the assembly of the complexes.  Ultimately, x-ray structure determinations are undertaken of these receptor complexes utilizing both in-house x-ray sources as well as the Stanford Synchrotron Radiation Laboratory (SSRL).

The dual approach of crystallographic studies on one hand, and functional studies on the other, allows us to reconcile the structural information with biological data.  Site-directed mutagenesis and receptor signaling assays are used to probe the functional effects of intermolecular contacts revealed by the crystal structures.  Engineered variants with interesting recognition and/or signaling properties will then be funneled into structural studies.  Efforts will also be made to discover novel receptor-binding molecules by structure-based modification of pre-existing ligands, computational search algorithms, phage display and synthetic chemical libraries.
 Recently, the laboratory has concentrated on biophysical studies of soluble extracellular domains of a number of signaling receptors for cytokines (gp130), peptide hormones (Atrial Natriuretic Factor), and antigen recognition (MHC & T cell receptor).  These systems have been directly implicated in cancer (gp130), cardiovascular disease (ANP receptor) and autoimmunity (TCR/MHC).  By expressing receptor domains truncated in such a way that a soluble fragment recapitulates the binding properties of the receptor in its natural environment, we have elucidated a number of novel paradigms for receptor recognition and, by inference, the subsequent activation mechanisms.   This strategy has been very successful for deciphering the recognition and activation mechanisms of membrane proteins which contain a single transmembrane (TM) helix linking extra- and intracellular domains.  For instance, while gp130 activation appears to require a sequential clustering of multiple receptor components into a hetero-oligomeric assembly, the ANP receptor extracellular domain undergoes a large conformational change which is likely propagated to the intracellular domains for triggering of an enzymatic activity.

 The limitation of our studies is that, until now we have only examined soluble domains.  For a full appreciation of the coupling of ligand recogntion and activation, a future direction of my laboratory is to determine structures of the entire receptor molecules so that both intra- and extracellular domains can be visualized simultaneously, along with the transmembrane region(s).