The X-ray Crystal Structure and Putative Ligand-derived Peptide Binding Properties of γ-Aminobutyric Acid Receptor Type A Receptor-associated Protein

David Knight,Richard Harris,Mark S.B Mcalister,John P Phelan,Stella Geddes,Stephen J Moss,Paul C Driscoll,Nicholas H Keep

doi:10.1074/jbc.m109753200

Abstract

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA(A) receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 A resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the gamma2 subunit of the GABA(A) receptor and the acidic carboxyl-terminal tails of human alpha- and beta-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.

Highlights

The ␥-aminobutyric acid receptor type A (GABAA) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABAA receptor and microtubules
We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the ␥2 subunit of the GABAA receptor and the acidic carboxylterminal tails of human ␣- and ␤-tubulin
There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification—For crystallography and later NMR samples, the GABARAP coding region was directionally cloned into a nonfusion derivative of expression vector pET28a (Novagen Inc., Stamford, CT), and the resulting construct was transformed into Escherichia coli BL21(DE3) cells grown at 37 °C in Luria Broth containing 30 mg/liter kanamycin. Earlier NMR samples were made using a pET-15b His-Tag construct This was grown under essentially the same conditions but with ampicillin (100 mg/liter) replacing the kanamycin. Crystallization and Data Collection—Recombinant GABARAP was crystallized at 22 °C using the hanging drop vapor diffusion technique by mixing 2 ␮l of protein at a concentration of 36 mg/ml in 10 mM Tris, 0.5 M NaCl (pH 7.0), and 2 ␮l of reservoir solution containing 0.1 M Tris (pH 8.5), 20% (w/v) polyethylane glycol monomethylether 2000, and 10 mM nickel(II) chloride hexahydrate. The crystals were in space group P212121 with a unit cell of a ϭ 30.16 Å, b ϭ 54.95 Å, and c ϭ 64.67 Å

No of atoms

RESULTS AND DISCUSSION

SRRRAGQKKL irrelevant