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Adiponectin is a highly abundant protein hormone secreted by adipose tissue. It elicits diverse biological responses, including anti-diabetic, anti-inflammatory, anti-tumor, and anti-atherosclerotic effects. Adiponectin consists of a globular domain and a collagen-like domain, and it occurs in three major oligomeric forms that self-assemble: trimers, hexamers, and high-molecular-weight oligomers. Adiponectin has been reported to bind to two seven-transmembrane domain receptors, AdipoR1 and AdipoR2, as well as to the protein T-cadherin, which is highly expressed in the cardiovascular system and binds only the high-molecular-weight form of adiponectin. The molecular mechanisms underlying this specificity remain unclear. Here we used a combination of X-ray crystallography and protein engineering to define the details of adiponectin's interaction with T-cadherin. We found that T-cadherin binds to the globular domain of adiponectin, relying on structural stabilization of this domain by bound metal ions. Moreover, we show that the adiponectin globular domain can be engineered to enhance its binding affinity for T-cadherin. These results help to define the molecular basis for the interaction between adiponectin and T-cadherin, and our engineered globular domain variants may be useful tools for further investigating adiponectin's functions.

G protein–coupled receptors (GPCRs), which mediate processes as diverse as olfaction and maintenance of metabolic homeostasis, have become the single most effective class of therapeutic drug targets. As a result, understanding the molecular basis for their activity is of paramount importance. Recent technological advances have made GPCR structural biology increasingly tractable, offering views of these receptors in unprecedented atomic detail. Structural and biophysical data have shown that GPCRs function as complex allosteric machines, communicating ligand-binding events through conformational change. Changes in receptor conformation lead to activation of effector proteins, such as G proteins and arrestins, which are themselves conformational switches. Here, we review how structural biology has illuminated the agonist-induced cascade of conformational changes that culminate in a cellular response to GPCR activation. Expected final online publication date for the Annual Review of Biophysics Volume 47 is M...

Camelid single-domain antibody fragments (‘nanobodies’) provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.