The Role of the β5-α11 Loop in the Active-site Dynamics of Acylated Penicillin-binding Protein A from Mycobacterium Tuberculosis

Overview

Journal J Mol Biol

Publisher Elsevier

Specialties Microbiology
Molecular Biology

Date 2012 Feb 28

PMID 22365933

Citations 9

Authors

Alena Fedarovich

Robert A Nicholas

Christopher Davies

Affiliations

Soon will be listed here.

Abstract

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.

Citing Articles

A conserved zinc-binding site in PBP2 required for elongasome-directed bacterial cell shape.

Micelli C, Dai Y, Raustad N, Isberg R, Dowson C, Lloyd A Proc Natl Acad Sci U S A. 2023; 120(8):e2215237120.

PMID: 36787358 PMC: 9974482. DOI: 10.1073/pnas.2215237120.

Recognition of the β-lactam carboxylate triggers acylation of penicillin-binding protein 2.

Singh A, Tomberg J, Nicholas R, Davies C J Biol Chem. 2019; 294(38):14020-14032.

PMID: 31362987 PMC: 6755799. DOI: 10.1074/jbc.RA119.009942.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from provide structural insights into β-lactam resistance.

Moon T, DAndrea E, Lee C, Soares A, Jakoncic J, Desbonnet C J Biol Chem. 2018; 293(48):18574-18584.

PMID: 30355734 PMC: 6290140. DOI: 10.1074/jbc.RA118.006052.

Crystal Structures of Penicillin-Binding Protein D2 from Listeria monocytogenes and Structural Basis for Antibiotic Specificity.

Jeong J, Cha H, Kim Y Antimicrob Agents Chemother. 2018; 62(9).

PMID: 30082290 PMC: 6125574. DOI: 10.1128/AAC.00796-18.

Have we realized the full potential of β-lactams for treating drug-resistant TB?.

Story-Roller E, Lamichhane G IUBMB Life. 2018; 70(9):881-888.

PMID: 29934998 PMC: 6119476. DOI: 10.1002/iub.1875.

References

Chen Y, Zhang W, Shi Q, Hesek D, Lee M, Mobashery S . Crystal structures of penicillin-binding protein 6 from Escherichia coli. J Am Chem Soc. 2009; 131(40):14345-54. PMC: 3697005. DOI: 10.1021/ja903773f. View

Veziris N, Truffot C, Mainardi J, Jarlier V . Activity of carbapenems combined with clavulanate against murine tuberculosis. Antimicrob Agents Chemother. 2011; 55(6):2597-600. PMC: 3101399. DOI: 10.1128/AAC.01824-10. View

Schuttelkopf A, van Aalten D . PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr. 2004; 60(Pt 8):1355-63. DOI: 10.1107/S0907444904011679. View

Otwinowski Z, Minor W . Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997; 276:307-26. DOI: 10.1016/S0076-6879(97)76066-X. View

Sainsbury S, Bird L, Rao V, Shepherd S, Stuart D, Hunter W . Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms. J Mol Biol. 2010; 405(1):173-84. PMC: 3025346. DOI: 10.1016/j.jmb.2010.10.024. View

Macheboeuf P, Di Guilmi A, Job V, Vernet T, Dideberg O, Dessen A . Active site restructuring regulates ligand recognition in class A penicillin-binding proteins. Proc Natl Acad Sci U S A. 2005; 102(3):577-82. PMC: 545533. DOI: 10.1073/pnas.0407186102. View

Jones T, Zou J, Cowan S, Kjeldgaard M . Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991; 47 ( Pt 2):110-9. DOI: 10.1107/s0108767390010224. View

Lim D, Strynadka N . Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat Struct Biol. 2002; 9(11):870-6. DOI: 10.1038/nsb858. View

Beadle B, Trehan I, Focia P, Shoichet B . Structural milestones in the reaction pathway of an amide hydrolase: substrate, acyl, and product complexes of cephalothin with AmpC beta-lactamase. Structure. 2002; 10(3):413-24. DOI: 10.1016/s0969-2126(02)00725-6. View

10.

Beadle B, Nicholas R, Shoichet B . Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation. Protein Sci. 2001; 10(6):1254-9. PMC: 2374021. DOI: 10.1110/ps.52001. View

11.

Jamin M, Wilkin J, Frere J . A new kinetic mechanism for the concomitant hydrolysis and transfer reactions catalyzed by bacterial DD-peptidases. Biochemistry. 1993; 32(28):7278-85. DOI: 10.1021/bi00079a026. View

12.

Lovering A, de Castro L, Lim D, Strynadka N . Structural analysis of an "open" form of PBP1B from Streptococcus pneumoniae. Protein Sci. 2006; 15(7):1701-9. PMC: 2242572. DOI: 10.1110/ps.062112106. View

13.

Flores A, Parsons L, Pavelka M . Genetic analysis of the beta-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to beta-lactam antibiotics. Microbiology (Reading). 2005; 151(Pt 2):521-532. DOI: 10.1099/mic.0.27629-0. View

14.

Dasgupta A, Datta P, Kundu M, Basu J . The serine/threonine kinase PknB of Mycobacterium tuberculosis phosphorylates PBPA, a penicillin-binding protein required for cell division. Microbiology (Reading). 2006; 152(Pt 2):493-504. DOI: 10.1099/mic.0.28630-0. View

15.

Dessen A, Mouz N, Gordon E, Hopkins J, Dideberg O . Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations. J Biol Chem. 2001; 276(48):45106-12. DOI: 10.1074/jbc.M107608200. View

16.

Fedarovich A, Nicholas R, Davies C . Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis. J Mol Biol. 2010; 398(1):54-65. PMC: 2854034. DOI: 10.1016/j.jmb.2010.02.046. View

17.

Sassetti C, Boyd D, Rubin E . Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol. 2003; 48(1):77-84. DOI: 10.1046/j.1365-2958.2003.03425.x. View

18.

Lavollay M, Arthur M, Fourgeaud M, Dubost L, Marie A, Veziris N . The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation. J Bacteriol. 2008; 190(12):4360-6. PMC: 2446752. DOI: 10.1128/JB.00239-08. View

19.

Chambers H, Turner J, Schecter G, Kawamura M, Hopewell P . Imipenem for treatment of tuberculosis in mice and humans. Antimicrob Agents Chemother. 2005; 49(7):2816-21. PMC: 1168716. DOI: 10.1128/AAC.49.7.2816-2821.2005. View

20.

Zhao G, Meier T, Kahl S, Gee K, Blaszczak L . BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins. Antimicrob Agents Chemother. 1999; 43(5):1124-8. PMC: 89121. DOI: 10.1128/AAC.43.5.1124. View