E. coli Molybdate-dependent transcriptional regulator (ModE)

George Ni '24 and Kelly Zhu '25

Contents:

I. Introduction

The Escherichia coli ModE (Molybdate-dependent transcription regulator) is the DNA binding protein that regulates the transcription of operons involved in uptake and utilization of molybdenum in E. coli. Molybdenum is the trace element that is significant for the redox reactions in E. coli, which acts as a catalyst in the process of nitrogen fixation. This element is bio-available for E. coli only in the form of molybdate (MoO42-) ModABCD operon is specifically responsible for coding high-affinity molybdate transport systems, and thus uptaking molybdate into the cell. In addition, ModABCD operon is negatively controlled by ModE.

As molybdate binds to ModE, the anion binding will cause a conformational change of ModE. It will then have a high affinity to bind to the site that is near to the promoter of ModABCD operon. ModE functions as a repressor that blocks the transcription.(Figure1) The higher intracellular level of molybdate increases the level of the conformational change of ModE, which in turn increases the binding affinity of ModE to ModABCD operon.

ModE
Figure.1 A schematic depiction of molybdate-dependent gene regulation in E.coli. Molybdate enters the bacterium via a high-affinity transporter system which comprises three proteins (ModA, ModB and ModC), and also, less efficiently, through the sulfate transporter which consists of CysP, T, W and A. Once in the cytoplasm the anion binds to the dimeric molybdate-dependent transcriptional activator (ModE), which in turn binds to and represses the modABCD operon. ModE also regulates the moaABCDE and dmsABC operons. Modified from Pau et al. (1998).

II. General Structure

ModE is a homodimer consisting of 262 amino acids. In each subunit, it contains N-terminal domains and C-terminal domains.
The N-terminal domain is mainly composed of alpha-helix (60% alpha-helix, 20% beta-strand) with 121 amino acid residues, and it contains a winged helix-turn helix motif. This motif is responsible for DNA binding and ModE dimerization.
The C-terminal domains is mainly composed of beta-sheet (25% alpha-helix, 60% beta-strand) with 141 amino acid residues. The C-terminal domains has two subunits (a and b).
Each subunit contains the Mop domain, which is responsible for binding with molybdate. 

III. Molybdate Binding

The DiMop dimer binds to two molybdate (MoO42-) by forming 9 hydrogen bonds with the charged oxygen atoms of molybdate. The interaction sites include residues of K183 , main chain amides of R128 and A184 ,side chain OH of s126 and s166 , OH and main chain amide of T163. The side chain of R128 also forms a salt bridge with the E218 ,which stabilizes the binding of molybdate by ModE.
The ligand-bound form of ModE then causes a conformational change of w186 , in which the side chain of tryptophan is more buried. This leads to an increase in the surface area of the dimerization interface of DiMop domain and thus a significant conformational change in the overall structure of ModE.

IV. DNA Binding

A After molybdate binds to ModE, the structural changes of the dimerization interface increase the affinity of ModE to the DNA molecule. In specific, the helix-turn-helix motif in the N-terminals of ModE will bind to the conserved DNA sequence 5'-CGTTATATTGTCGCCTACATAACG-3', which is the upstream DNA segments of ModABCD operon.
ModE first identifies the palindromic recognition site of the conserved sequence.(Figure2) Alpha3 of the helix-turn-helix motif then binds to the major groove of the DNA. The wing of the motif (amino acid residues 61-78) interacts with the adjacent minor groove. Lysines residues on the wing interact with the phosphate backbone of DNA. The binding lets ModE bend the DNA slightly toward itself, and thus inhibits the transcription of ModABCD operon.

DNA
Figure.2 A model of ModE interacting with double helix DNA. The DNA strands are purple and cyan, the palindromic DNA recognition site is black.Modified from Pau et al. (1998).


VI. References

Gourley, D. G., Schuttelkopf, A. W., Anderson, L. A., Price, N. C., Boxer, D. H., & Hunter, W. N. (2001). Oxyanion Binding Alters Conformation and Quaternary Structure of the C-terminal Domain of the Transcriptional Regulator ModE: IMPLICATIONS FOR MOLYBDATE-DEPENDENT REGULATION, SIGNALING, STORAGE, AND TRANSPORT*. Journal of Biological Chemistry, 276(23), 20641-20647. https://doi.org/10.1074/jbc.M100919200

Hall, D. R., Gourley, D. G., Leonard, G. A., Duke, E. M. H., Anderson, L. A., Boxer, D. H., & Hunter, W. N. (1999). The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds. The EMBO Journal, 18(6), 1435-1446. https://doi.org/10.1093/emboj/18.6.1435

Schuttelkopf, A. W., Boxer, D. H., & Hunter, W. N. (2003). Crystal Structure of Activated ModE Reveals Conformational Changes Involving Both Oxyanion and DNA-binding Domains. Journal of Molecular Biology, 326(3), 761-767. https://doi.org/10.1016/S0022-2836(02)01358-X

Walkenhorst, H. M., Hemschemeier, S. K., & Eichenlaub, R. (1995). Molecular analysis of the molybdate uptake operon, modABCD, of Escherichia coli and modR, a regulatory gene. Microbiological Research, 150(4), 347-361. https://doi.org/10.1016/S0944-5013(11)80016-9

Bank, R. P. D. (n.d.). 3D View: 1B9N. REGULATOR FROM ESCHERICHIA COLI. Retrieved December 9, 2022, from https://www.rcsb.org/3d-view/1B9N

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