CryoEM structure of Rv2531c reveals cofactor-induced tetramer-dimer transition in a tuberculin amino acid decarboxylase

Abstract

The survival of Mycobacteriumtuberculosis relies on its ability to adapt to dynamic and hostile host environments. Amino acid decarboxylases play a crucial role in these adaptations, but their structural and mechanistic properties are not fully understood. Bioinformatic analyses revealed that these enzymes exist in three distinct forms based on their domain organization. We used cryoEM at 2.76 Å resolution to show that Rv2531c exhibits unexpected oligomeric and conformational flexibility. The enzyme forms a tetramer with distinct open and closed conformations in its apo state, suggesting dynamic intersubunit interactions. Upon binding pyridoxal 5'-phosphate, the enzyme undergoes a dramatic structural rearrangement, transitioning into a dimer. These findings reveal a novel mechanism of oligomeric plasticity. We also uncover an amino-terminal domain that might play a role in this process. Our results provide critical insights into the structural adaptations that support bacterial persistence under intracellular stress. By elucidating the apo and pyridoxal 5'-phosphate-bound states of Rv2531c, we contribute to a deeper understanding of how M. tuberculosis navigates its challenging intracellular environment. These insights into the unique structural features of Rv2531c offer a foundation for targeting metabolic resilience in tuberculosis and open avenues for future studies on the role of this domain in pathogenesis.

Keywords: Mycobacterium tuberculosis; cryogenic electron microscopy; glutamate decarboxylase; pyridoxal 5'-phosphate; γ-aminobutyric acid.

Figure 1

Domain organization and oligomerization of Rv2531c.A, amino-terminal domain (NTD; residues 1–131), response regulator domain (RRD; 134–288) aka receiver domain, catalytic domain (291–741), carboxy-terminal domain (CTD; 748–947). Mycobacterium abscessus has no equivalent NTD, and Escherichia coli only has the catalytic and carboxy-terminal domains. B, size-exclusion chromatogram (SEC; pH 7.5) of the apo tetramer (red), which elutes as a tetramer and the PLP-bound Rv2531c (blue), which elutes as a dimer. C, apo Rv2531c elutes as a tetramer at pH 8.5 (top) or 7.5 (bottom). D, SDS-PAGE of SEC fractions from the chromatogram at the bottom of C (at pH 7.5). Lane 1, molecular weight marker; 2, nickel–nitrilotriacetic acid fraction prior to SEC; 3 to 5, void peak fractions; and 6 to 13, peak fractions. PLP, pyridoxal 5′-phosphate.

Figure 2

CryoEM structure of the PLP-bound Rv2531c dimer.A, cartoon drawing of the PLP-bound dimer. PLP is shown as spheres. Each Rv2531c protomer is colored spectrally (NTD, residues 1–131, green; RRD, 134–288, orange; catalytic domain [CD], 291–741, magenta; and CTD, 748–947, blue). B, cryoEM map of the Rv2531c dimer with C2 symmetry, front and back view as indicated. Each domain is colored differently. C, interface residues within the PLP-linked Rv2531c dimer. The catalytic domain of one protomer is colored purple. The other protomer is colored gray. CTD, carboxy-terminal domain; NTD, amino-terminal domain; PLP, pyridoxal 5′-phosphate; RRD, response regulator domain.

Figure 3

CryoEM Rv2531c structure.A, cartoon drawing of the PLP-bound Rv2531c protomer. The four α-helices of the amino-terminal domain (NTD, colored in green) are labeled. The following response regulator domain (RRD) is shown in orange. The catalytic domain is colored and labeled in magenta. The carboxy-terminal domain (CTD) is shown in blue. B, Coulomb potential map of the PLP binding site. C, predicted binding mode of glutamate to the Rv253c1 dimer, obtained through molecular docking using AutoDock Vina within ChimeraX (36, 42). Glutamate and PLP are shown in gray; the catalytic domain of Rv253c1 is depicted in magenta, and the carboxy-terminal domain in blue. The most favorable binding conformation, exhibiting a docking score of −4.5, is shown. PLP, pyridoxal 5′-phosphate.

Figure 4

Rv2531c has two stable tetrameric configurations. View of Rv2531c as an open (A) or a closed (B) tetramer. Each domain is colored differently (NTD, residues 1-131, green; RRD, 134-288, orange; catalytic domain, 291-741, magenta; and CTD, 748-947, blue). Top row, Coulomb potential maps. Bottom row, two opposite residing protomers showing all bonds. A, the open tetramer measures about 145 Å by 113 Å by 77 Å. B, the closed tetramer measures about 129 Å by 120 Å by 77 Å. CTD, carboxy-terminal domain; NTD, amino-terminal domain; RRD, response regulator domain.

Figure 5

Distinct protomer arrangements determine the oligomeric Rv2531c architecture. Residues 291 to 947 are shown in the same orientation in all panels. The catalytic and carboxy (CTD) terminal domains of the protomer of our dimeric (A and B) and tetrameric open (C) and closed (D) structures are colored spectrally from green to red as indicated. Residues 291 to 947 are almost identical in all three structures, with RMSD of 1.322 Å for 6511 atoms for the protomer of the open or closed (RMSD of 1.556 Å for 6558 atoms) tetramer relative to the protomer in the PLP-bound dimer shown in B. However, the amino-terminal domain (NTD, residues 1–131) and the response regulator domain (RRD; 134–288; blue), aka receiver domain, are pivoted toward one side by about 100° in (C) the open tetramer structure, whereas they are rotated in (D) the other direction by about −80° in the closed tetramer structure. PLP, pyridoxal 5′-phosphate.