Molecular Pharmacology of P2X Receptors: Exploring Druggable Domains Revealed by Structural Biology.

Extracellular ATP is a critical signaling molecule that is found in a wide range of concentrations across cellular environments. The family of nonselective cation channels that sense extracellular ATP, termed P2X receptors (P2XRs), is composed of seven subtypes (P2X1-P2X7) that assemble as functional homotrimeric and heterotrimeric ion channels. Each P2XR is activated by a distinct concentration of extracellular ATP, spanning from high nanomolar to low millimolar. P2XRs are implicated in a variety of physiological and pathophysiological processes in the cardiovascular, immune, and central nervous systems, corresponding to the spatiotemporal expression, regulation, and activation of each subtype. The therapeutic potential of P2XRs is an emerging area of research in which structural biology has seemingly exceeded medicinal chemistry, as there are several published P2XR structures but currently no FDA-approved drugs targeting these ion channels. Cryogenic electron microscopy is ideally suited to facilitate structure-based drug design for P2XRs by revealing and characterizing novel ligand-binding sites. This review covers structural elements in P2XRs including the extracellular orthosteric ATP-binding site, extracellular allosteric modulator sites, channel pore, and cytoplasmic substructures, with an emphasis on potential therapeutic ligand development.

The 2.2 A resolution crystal structure of Bacillus cereus Nif3-family protein YqfO reveals a conserved dimetal-binding motif and a regulatory domain.

YqfO of Bacillus cereus is a member of the widespread Nif3 family of proteins, which has been highlighted as an important target for structural genomics. The N- and C-terminal domains are conserved across the family and contain a dimetal-binding motif in a putative active site. YqfO contains an insert in the middle of the protein, present in a minority of bacterial family members. The structure of YqfO was determined at a resolution of 2.2 A and reveals conservation of the putative active site. It also reveals the previously unknown structure of the insert, which despite extremely limited sequence conservation, bears great similarity to PII, CutA, and a number of other trimeric regulatory proteins. Our results suggest that this domain acts as a signal sensor to regulate the still-unknown catalytic activity of the more-conserved domains.

The Sampling of Conformational Dynamics in Ambient-Temperature Crystal Structures of Arginine Kinase.

Arginine kinase provides a model for functional dynamics, studied through crystallography, enzymology, and nuclear magnetic resonance. Structures are now solved, at ambient temperature, for the transition state analog (TSA) complex. Analysis of quasi-rigid sub-domain displacements show that differences between the two TSA structures average about 5% of changes between substrate-free and TSA forms, and they are nearly co-linear. Small backbone hinge rotations map to sites that also flex on substrate binding. Anisotropic atomic displacement parameters (ADPs) are refined using rigid-body TLS constraints. Consistency between crystal forms shows that they reflect intrinsic molecular properties more than crystal lattice effects. In many regions, the favored directions of thermal/static displacement are appreciably correlated with movements on substrate binding. Correlation between ADPs and larger substrate-associated movements implies that the latter approximately follow paths of low-energy intrinsic motions.

Structural basis for the preference of UTP over ATP in human deoxycytidine kinase: illuminating the role of main-chain reorganization.

Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65-79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240-247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK.