Chlamydia trachomatis glyceraldehyde 3-phosphate dehydrogenase: Enzyme kinetics, high-resolution crystal structure, and plasminogen binding

Abstract

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an evolutionarily conserved essential enzyme in the glycolytic pathway. GAPDH is also involved in a wide spectrum of non-catalytic cellular 'moonlighting' functions. Bacterial surface-associated GAPDHs engage in many host interactions that aid in colonization, pathogenesis, and virulence. We have structurally and functionally characterized the recombinant GAPDH of the obligate intracellular bacteria Chlamydia trachomatis, the leading cause of sexually transmitted bacterial and ocular infections. Contrary to earlier speculations, recent data confirm the presence of glucose-catabolizing enzymes including GAPDH in both stages of the biphasic life cycle of the bacterium. The high-resolution crystal structure described here provides a close-up view of the enzyme's active site and surface topology and reveals two chemically modified cysteine residues. Moreover, we show for the first time that purified C. trachomatis GAPDH binds to human plasminogen and plasmin. Based on the versatility of GAPDH's functions, data presented here emphasize the need for investigating the Chlamydiae GAPDH's involvement in biological functions beyond energy metabolism.

Keywords: Chlamydia; GAPDH; STD/STI; crystal structure; enzyme kinetics; glycolysis; plasmin binding; plasminogen binding; protein-protein interaction; reactive cysteine.

FIGURE 1

Determination of pH optimum and kinetic parameters of CtGAPDH enzyme activity. (a) CtGAPDH activity is shown at different pH. Initial reaction velocity (v₀) was measured. (b) K _M and V _max for NAD⁺ were determined from M‐M plot. Assays were done in triplicate. (c) K _M and V _max for D‐G3P were determined from M‐M plot. Assays were done in triplicate

FIGURE 2

Structure of CtGAPDH monomer and the assembly into dimers and tetramer. (a) Cartoon drawing showing the tetrameric assembly of CtGAPDH. NAD⁺ is shown in stick model. Major interface axes P and Q are labeled. S‐loop is colored fire brick. (b) Subunits A, B and C, D form dimers. The AB dimer is shown with a close‐up view of the major interface highlighted in green color on subunit A. (c) Subunits A and C assemble across the Q‐axis. Interface area on subunit A is shown in magenta color. The S‐loop in subunit C is shown in fire brick color. (d) The catalytic domain and the NAD⁺‐binding domain in subunit A are colored yellow and light orange, respectively. Dimer interface area is shown in green. The S‐loop is in fire brick color. The S‐loop is also part of the second largest interface. Areas with strictly conserved residues (7–13 and 145–154) near the NAD⁺‐binding pocket are shown in magenta color. NAD⁺ is shown in stick model

FIGURE 3

Interactions between NAD⁺ and CtGAPDH, and comparison of active sites in the binary and ternary complex. (a) Interactions between CtGAPDH (subunit A) residues and NAD⁺. CtGAPDH residues (C: yellow) and NAD⁺ (C: green) are shown in stick models. Hydrogen bonding distances (Å) are shown. (b) Superposition of subunits A of CtGAPDH and GBSGAPDH ternary complex (5JYA). NAD⁺ and the substrate in 5JYA, and the catalytic residues Cys150 and His177 in CtGAPDH are in stick model (C: wheat color). The distance between Cys150Sγ and the C1 atom of the substrate is 3.6 Å, and between Cys150Sγ and His177Nε it is 3.3 Å. The catalytic residues of 5JYA are also shown in stick model (C: slate color). The catalytic cysteine was mutated to serine in 5JYA. Areas where the conformations differ significantly are shown in dark blue on the GBSGAPDH structures and the CtGAPDH residues bordering the divergent areas are labeled. One water molecule (W177 shown as magenta sphere) is found at or near the site for the O3P atom of the substrate

FIGURE 4

Electron density for the modified residues CME287 and SNC63, and their environment in CtGAPDH crystal structure. (a) Fo‐Fc omit maps (mesh in light pink contoured at 3σ) for CME287 (upper panel) and SNC63 (lower panel). The residues are shown in stick model (C: white, N: blue, O: red, S: yellow). (b) Aromatic and hydrophobic residues surrounding CME287 (C: green) are shown in stick model (C: yellow). (c) Stereo diagram showing aromatic and basic residues within 5 Å of SNC63 (stick model, C: white)

FIGURE 5

Comparison of different areas where CtGAPDH sequence diverges from human and GBS GAPDH. (a) Cartoon drawing showing superposition of subunit A of CtGAPDH (light yellow) and hGAPDH (1U8F subunit O, light pink). Regions where the sequence identities are low are marked and shown in purple blue, slate and marine color. Comparison of these regions in the sequences for Ct and human (Hs) GAPDHs are shown. NAD⁺ is shown in stick model (C: green). (b) Close‐up view of the adenine binding pocket. Residues in hGAPDH (C: orange), CtGAPDH (C: yellow) and NAD⁺ (C: magenta and green in hGAPDH and CtGAPDH respectively) are shown in stick models. (c) Superposition of subunit A of CtGAPDH (light yellow) and GBSGAPDH (5JYA subunit A, grey). Regions 2 and 3 where the sequence identities are low are marked and shown in slate and marine color. Comparison of these regions in the sequences for CtGAPDH and GBSGAPDH are shown. NAD⁺ is shown in stick model (C: green)