Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Abstract

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein.A structure was also obtained where glyceraldehyde 3-phosphate binds in the P s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of ␣-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS 3 in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1-3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6 -8). Investigations showed that treatment of sperm with ␣-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9 -11), sperm motility (9 -13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15-17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18,19), pro...

High‐resolution crystal structures of the photoreceptor glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) with three and four‐bound NAD molecules

“…Residues located in disallowed regions in our structures were found within similar regions in many other GAPDH structures. [19][20][21] Water molecules were added to the present models with Coot. The peak contoured at 1.8r in 2Fo-Fc maps was identified as a water molecule.…”

“…GAPDH enzymatic activity requires the binding of NAD but the enzyme shows different binding characteristics depending on the tissue source. [19][20][21][22][23] Here, we present two high resolution crystal structures of GAPDH from ROS with different occupancies of NAD. These GAPDH structures provide an updated view of their NAD-binding sites, and their conformational changes induced by NAD binding.…”

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

“…When typical E. coli strains were used for expression, mass spectrometry analyses confirmed that mouse GAPDHS and the host bacterial GAPDH formed mixed tetramers, resulting in the co-purification of bacterial and recombinant enzymes (data not shown). A similar observation has been made for the rat GAPDHS expressed in E. coli with the purified tetrameric enzyme consisting of 3 subunits of E. coli GAPDH and 1 subunit of rat GAPDHS [7]. Our approach was to express recombinant human GAPDHS lacking the proline-rich N-terminal region in a GAPDH-deficient E. coli strain (CGSC strain #7563).…”

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is expressed at high yield as an active homotetramer in baculovirus-infected insect cells

“…The expression of recombinant GAPDH enzymes in E. coli has met with varied success, with high production of insoluble material frequently observed [9]. A recent study found that expression of rat GAPDHS, either as a full-length protein or a truncated protein lacking the proline-rich N-terminus, resulted in protein that was predominantly insoluble [5]. We have had similar difficulties obtaining high yields of soluble protein when expressing recombinant mouse or human GAPDHS in E. coli.…”

“…We have had similar difficulties obtaining high yields of soluble protein when expressing recombinant mouse or human GAPDHS in E. coli. Although native GAPDH enzymes typically function as homotetramers (molecular mass ~150 kDa), the expression of rat GAPDHS in E. coli resulted in mixed tetramers consisting of 1 subunit of rat GAPDHS and 3 subunits of E. coli GAPDH [5]. The generation of GAPDH heterotetrameric forms combining purified GAPDH dimers from two different species has been reported [10–11].…”