PNNL

Abstract

C2 domains are ubiquitous membrane-binding modules of about 130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a C-terminal XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it may be important. To determine if the C2 domains associated with XYPPX-repeat domains behave like typical C2 domains, the C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and functionally characterized using X-ray crystallography and NMR spectroscopy. Crystal structures for Tv-C2-1 were solved with and without bound sulfate ligands at a resolution of 1.73 and 1. 85 Å, respectively. The domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel ?-strands in the type-2 topology. All 123 native backbone amide cross peaks were observed and assigned in the 1H-15N HSQC spectrum of Tv-C2-1 along with 94 % of the backbone carbonyl chemical shifts and most of the side chain carbon and proton chemical shifts. An NMR chemical shift perturbation study with Ca2+ showed that Tv-C2-1 bound two Ca2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with Kds of 58.0 ± 0.1 ?M and 232 ± 6 ?M. Estimations of the overall rotational correlation time, ?c, in the apo- (10.8 ns) and Ca2+ bound (9.5 ns) state suggests the protein becomes more compact upon Ca2+ binding, consistent with a decrease in dynamics in loop3 and (marginally) loop-1 suggested by amide 15N heteronuclear steady-state {1H}-15N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted.