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Title: Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA]

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimatedmore » dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less
Authors:
ORCiD logo [1] ;  [2] ;  [3] ;  [4] ;  [5] ; ORCiD logo [6] ;  [7] ; ORCiD logo [6] ;  [2] ; ORCiD logo [6]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States); Genentech Inc., South San Francisco, CA (United States)
  4. Univ. of California, Berkeley, CA (United States); Univ. of Hawaii-Manoa, Honolulu, HI (United States)
  5. Univ. of California, Berkeley, CA (United States)
  6. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  7. Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Univ. of Washington, Seattle, WA (United States); Center for Infectious Disease Research, Seattle, WA (United States)
Publication Date:
Report Number(s):
LA-UR-17-24508
Journal ID: ISSN 0006-2960
Grant/Contract Number:
AC52-06NA25396
Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 56; Journal Issue: 30; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
National Institutes of Health (NIH); USDOE
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; Biological Science; tuberculosis; Ser/Thr protein kinases; virulence factors; host-pathogen interactions; toll-like receptor 2 activation
OSTI Identifier:
1411356