A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion

Rodríguez-Hernández Annia,Enrique Romo-Arévalo,Adela Rodríguez-Romero

doi:10.3390/cryst9120622

Rodríguez-Hernández Annia, Enrique Romo-Arévalo + Show 1 more

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https://doi.org/10.3390/cryst9120622

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Journal: Crystals	Publication Date: Nov 26, 2019
Citations: 2	License type: CC BY 4.0

Affiliation: Universidad Nacional Autónoma de México

Abstract

Escherichia coli (E. coli), one of the most frequently used host for the expression of recombinant proteins, is often affected by the toxic effect of the exogenous proteins that is required to express. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multi-functional protein that has been used as a control marker for basal function and it is known to undergo cysteine oxidation under different types of cellular stress. Here, we report the 3D structure of the endogenous GAPDH purified from stressed E. coli cells expressing a eukaryotic protein. The structure was solved at 1.64 Å using single-wavelength anomalous dispersion (SAD) phasing with a selenium-modified enzyme. Interestingly, each GAPDH monomer contains a molecule of glyceraldehyde-3 phosphate in a non-previously identified site. Furthermore, the catalytic Cys149 is covalently attached to a ~300 Da molecule, possibly glutathione. This modification alters the conformation of an adjacent alpha helix in the catalytic domain, right opposite to the NAD+ binding site. The conformation of the alpha helix is stabilized after soaking the crystals with NAD+. These results exemplify the effects that the overexpression of an exogenous protein has over the host proteins and sheds light on the structural changes that large oxidant molecules on the catalytic cysteine produce for the GAPDH enzyme.

Highlights

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, highly studied enzyme that reversibly transforms glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate in the presence of NAD+ and inorganic phosphate in the glycolysis pathway [1]
Once that we identified GAPDH in the Se-modified crystal and obtained a refined model, we determined the three-dimensional structure of E. coli GAPDH (EcGAPDH) from the native and NAD+ crystals
The crystals soaked in NAD+ showed a significant reduction in the electron density surrounding Cys149; in this case, the residue was modeled as a cysteine sufinic acid (Figure 4b). These results suggest that the presence of the exogenic plasmid or the expression of the human protein could have produced the oxidation of the Cys149; the stress caused by the use of minimum media, increased the number of modified EcGAPDH, this data is in agreement with the higher amount of stress observed in E. coli during the expression of recombinant proteins [37]

Summary

Introduction

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, highly studied enzyme that reversibly transforms glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate in the presence of NAD+ and inorganic phosphate in the glycolysis pathway [1]. Apart from its glycolytic function, GAPDH displays a battery of moonlighting activities. In eukaryotic organisms, it participates in membrane transport, DNA repair and replication, autophagy, pathogenesis, host immunosuppression, and microtubule building, among others [4,5,6,7,8]. Several protein-binding partners have been identified for GAPDH, suggesting its participation in a variety of non-canonical functions, similar to the ones described in eukaryotes [9].

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