Poster abstracts

Poster number 65 submitted by Xin Li

Structure of the Nitrosomonas europaea Rh protein

Xin Li (The Ohio State Biophysics Program, and Departments of Biochemistry and Chemistry, The Ohio State University), Sanjay Jayachandran (TransMembrane Biosciences, 145 N. Sierra Madre, Suite #5, Pasadena, CA 91107), Hiep-Hoa Nguyen (TransMembrane Biosciences, 145 N. Sierra Madre, Suite #5, Pasadena, CA 91107), Michael K. Chan (The Ohio State Biophysics Program, and Departments of Biochemistry and Chemistry, The Ohio State University)

Abstract:
Amt/Rh proteins are a superfamily of integral membrane proteins. While Amt proteins are agreed to transport ammonia (NH₃/NH₄⁺), the biological function of Rh proteins has been controversial. Proposed roles as an ammonia transporter, a CO₂ transporter and/or a structural protein involved in stabilizing cell skeleton of red blood cells have all been suggested. Given the clear phylogenetic differences between Rh and Amt proteins, a structural comparison of the two families could be illuminating. Although there have now been a number of structures of Amt proteins, structures of Rh proteins have lagged. We use crystallography to study the structure of Rh proteins. We recently improved the crystal diffraction resolution to 1.85 Å and obtained Se-Met phases that allowed us to determine the structure. This is the first structure of an Rh family member, the Rh protein from the chemolithoautotrophic ammonia-oxidizing bacterium Nitrosomonas europaea. This Rh protein exhibits a number of similarities to its Amt cousins, including a trimeric oligomeric state, a central pore with an unusual twin-His site in the middle, and a Phe residue that blocks the channel for small molecule transport. However, there are some significant differences, the most notable being the presence of an additional cytoplasmic C-terminal α helix, an increased number of internal proline residues along the transmembrane helices, as well as a specific set of residues that appear to link the C-terminal helix to the Phe blockage. This latter linkage suggests a mechanism in which binding of a partner protein to the C-terminus could regulate channel opening. Another difference is the absence of the extracellular π-cation binding site conserved in Amt structures. Instead, CO₂ pressurization experiments identify a CO₂ binding site near the intracellular exit of the channel whose residues are highly conserved in all Rh proteins, except those belonging to the Rh30 subfamily. The structure of the N. europaea Rh protein provides a framework to elucidate the molecular mechanism of Rh protein family. The discovery of C-terminal tail sheds light on verifying Rh protein physiological functions by identifying its binding partners.

References:
Li,X., Jayachandran,S., Nguyen,H.H., and Chan,M.K. 2007. Structure of the Nitrosomonas europaea Rh protein. Proc Natl Acad Sci U S A 104:19279-19284.

Keywords: channel, dioxide, proline kinkscarbon