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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.

SUBMITTER: Schormann N

PROVIDER: S-EPMC7679969 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Schormann Norbert N Campos Juan J Motamed Rachael R Hayden Katherine L KL Gould Joseph R JR Green Todd J TJ Senkovich Olga O Banerjee Surajit S Ulett Glen C GC Chattopadhyay Debasish D

Protein science : a publication of the Protein Society 20201030 12

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 s ...[more]

PMID: 33058314

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Project description:BACKGROUND: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. RESULTS: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2A resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. CONCLUSION: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.

Project description:The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an "intrinsic" cell-autonomous program inherent to each RB and an "extrinsic" environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB.IMPORTANCEChlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium's pathogenesis.

Dataset Information

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

Publications

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

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