The Crystal and Solution Structures of Glyceraldehyde-3-phosphate Dehydrogenase Reveal Different Quaternary Structures

Pedro J.B. Pereira,Dmitri I. Svergun,Manfred Roessle,Ana M. Damas,Frederico Ferreira-da-Silva,Pedro Moradas-Ferreira,Luís Gales

doi:10.1074/jbc.m605267200

Abstract

The presence of an isoform of glyceraldehyde-3-phosphate dehydrogenase (kmGAPDH1p) associated with the cell wall of a flocculent strain of Kluyveromyces marxianus was the first report of a non-cytosolic localization of a glycolytic enzyme, but the mechanism by which the protein is transported to the cell surface is not known. To identify structural features that could account for the multiple localizations of the protein, the three-dimensional structure of kmGAPDH1p was determined by x-ray crystallography and small angle x-ray scattering. The x-ray crystallographic structure of kmGAPDH1p revealed a dimer, although all GAPDH homologs studied thus far have a tetrameric structure with 222 symmetry. Interestingly, the structure of kmGAPDH1p in solution revealed a tetramer with a 70 degrees tilt angle between the dimers. Moreover, the separation between the centers of the dimers composing the kmGAPDH1p tetramer diminished from 34 to 30 A upon NAD(+) binding, this latter value being similar to the observed in the crystallographic models of GAPDH homologs. The less compact structure of apo-kmGAPDH1p could already be the first image of the transition intermediate between the tetramer observed in solution and the dimeric form found in the crystal structure, which we postulate to exist in vivo because of the protein's multiple subcellular localizations in this yeast species.

Highlights

Regional (FEDER) and Fundacao para a Ciencia e a Tecnologia (FCT), Portugal (Project POCI/SAU-NEU/58735/2004)
The unexpected presence of a GAPDH isoform at the cell wall of K. marxianus led us to investigate the three-dimensional structure of the protein
KmGAPDH1p is detected at the cell surface of flocculent K. marxianus cells, it does not contain an N-terminal endoplasmic reticulum (ER)-targeting peptide [36]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—kmGAPDH1 open reading frame was amplified by PCR and cloned into pQE32 (Qiagen). The cell pellet was resuspended in buffer A (300 mM NaCl, 10 mM imidazole, 1 mM ␤-mercaptoethanol, 1 mg/ml lysozyme, 10 mM Tris-HCl, pH 7.5). A washing step was carried out with buffer B (1 M NaCl, 20 mM imidazole, 1 mM ␤-mercaptoethanol, 10 mM Tris-HCl, pH 7.5) until the A280 nm value remained invariant (10 column volumes). KmGAPDH1p-containing fractions were pooled and dialyzed against buffer C (150 mM NaCl, 1 mM ␤-mercaptoethanol, 1 mM EDTA, 10 mM TrisHCl, pH 7.5), concentrated to 13 mg/ml, frozen in liquid nitrogen, and stored at Ϫ70 °C. Structure Determination and Refinement—The structure was solved by molecular replacement using the program AMoRe [43] and the coordinates of E. coli GAPDH (Protein Data Bank code 1GAD [1]) as search model.

Structure refinement

RESULTS

DISCUSSION