How Do Gepotidacin and Zoliflodacin Stabilize DNA Cleavage Complexes with Bacterial Type IIA Topoisomerases? 1. Experimental Definition of Metal Binding Sites
Abstract
:1. Introduction
No. Ref. | PDB Code + Res in Å | Inhibitor Type | DNA Sequence Central Six Base-Pairs 1 | Inhibitor Pockets Occupied 2 | Catal. Tyr 123 3 | |||||
---|---|---|---|---|---|---|---|---|---|---|
1 | 1′ | 2D | 2A | 3 | 3′ | |||||
1 [11] | 2xcs-v2-BA-x.pdb* 2.1 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
2 [27] | 4bul-BA-x.pdb 2.6 Å | NBTI | 5′ TGTACC 3′ 3′ ACATGG 5′ | - | - | x | x | - | - | Phe |
3 [29] | 4plb 2.69 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
4 [28] | 5bs3 2.65 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
5 [9] | 5cdm-v2-BA-x.pdb* 2.5 Å | SPT | 5′ C-YGGCCG 3′ | x | x | - | - | - | - | TyrP |
6 [9] | 5cdp-BA-x.pdb 2.45 Å | Etop. | 5′ G-PGTACC 3′ | x | - | - | - | - | - | Phe |
7 [9] | 5cdr-v2-BA-x.pdb* 2.65 Å | - | 5′ G-PGTACC 3′ | - | - | - | - | - | - | Phe |
8 [26] | 5iwi-v2-BA-x.pdb* 1.98 Å | NBTI | 5′ GGTAC A 3′ 3′ CCATG-T 5′ | - | - | x | x | - | - | Phe |
9 [26] | 5cdp-BA-x.pdb 2.5 Å | NBTI | 5′ GGTTCA 3′ 3′ CCAAGT 5′ | - | - | x | x | - | - | Phe |
10 [39] | 5npp-BA-x.pdb 2.22 Å | NBTI + thiop. | 5′ G-PGTACC 3′ | - | - | x | x | x | x | Tyr |
11 [30] | 6fm4 2.7 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
12 [33] | 6fqs-BA-x.pdb 3.11 Å | IPY | 5′ C-YGGCCG 3′ | x | x | - | - | - | - | TyrP |
13 [6] | 6qtk-BA-x.pdb 2.31 Å | NBTI | 5′ G-PGTACC 3′ | - | - | x | x | - | - | Phe |
14 [40] | 6qx1-BA-x.pdb 2.65 Å | benzoi’ | 5′ G-PGTACC 3′ | - | - | - | - | x | x | Phe |
15 [41] | 6z1a 2.3 Å | NBTI | 5′ G-PGTACC 3′ | - | - | x | x | - | - | Phe |
16 [31] | 7fvs 2.16 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
17 [31] | 7fvt 2.08 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
18 [32] | 7mvs 2.60 Å | NBTI | 5′ GGGCCC 3′ | - | - | x | x | - | - | Phe |
19 [8] | 8bp2-BA-x.pdb 2.80 Å | SPT | 5′ C-YGGCCG 3′ | x | x | - | - | - | - | TyrP |
20 t.p. | 9fz6-BA-x.pdb 2.58 Å | - | 5′ G-PGTACC 3′ | - | - | - | - | - | - | Phe |
2. Results
2.1. High-Resolution Structures Have Clearly Defined Metals at Either the 3′(A)-Site or Y(B)-Site
2.2. A Comparison of the Original 3l4k Structure Interpreted as Having the Same Metal Ion Coordination Geometry Independent of the Active Sites with Re-Refined Coordinates
2.3. Re-Refined P61 S. aureus Structures Suggest a Manganese Ion and BisTris Mediate an Important Crystal Contact
2.4. An Anomalous Dataset Confirmed Catalytic Metal and Crystal Contact Manganese Ions, but Unexpectedly Showed Additional Manganese Density near Serine 84
Mn Site | Occupancy | Peak Height (rmsd) |
---|---|---|
B/5081 | 1.0 | 15.5 |
D/5081 | 1.0 | 15.1 |
E/1999 | 0.6 | 7.0 |
F/1999 | 0.5 | 4.3 |
C/901 | 0.2 | 4.0 |
3. Discussion
4. Materials and Methods
4.1. The Standard BA-x Nomenclature for S. aureus DNA Gyrase Crystal Structures and the Y(B), 3′(A) Nomenclature for Catalytic Metals
4.2. Re-Refining Fully and Partially Occupied Metal Binding Sites with Chemically Reasonable Geometry
4.3. Protein Purification and Crystallization of a DNA Gyrase—DNA Complex
4.4. Data Collection, Structure Determination and Refinement
4.5. The Generation of a Movie of Geoptidacin Binding
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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PDB | Compound | Resol. (Å) | Crystallization Buffer pH | Mn C901 Occupancy (Bfactor) | His C390 Side-Chain Bfactor |
---|---|---|---|---|---|
2xcs-v2-BA-x.pdb | GSK299423 | 2.10 | 6.5 | 0.65 (23.3) | 23.7 |
5iwi-v2-BA-x.pdb * | GSK945237 | 1.98 | 6.2 | 0.30 (35.8) | 35.5 |
5cdm-v2-BA-x.pdb | QPT-1 | 2.50 | 6.2 | 0.40 (55.2) | 52.1 ** |
5cdr-v2-BA-x.pdb | - | 2.65 | 6.2 | 0.30 (37.5) | 45.8 |
Mechanism Number and Name | Problems with Mechanism (Current Estimate of Mechanism Being Broadly Correct). Reference. |
---|---|
(i) a two-metal mechanism from 2010 | No chemically sensible structure has yet been deposited in support of this mechanism. The X-ray data are consistent with a single moving metal mechanism (see Figure 3). The topo1A lysine is also consistent with the single moving metal mechanism in [50]. Some variation on this two-metal mechanism cannot be entirely excluded: (Low to very low) Schmidt et al. (2010) [43]. |
(ii) Single-metal mechanism from 2019 | The mechanism proposed does not detail any sensible chemistry. The mechanism seems to propose that the metal starts at the A-site and cleaves the DNA before moving to the B-site. Some variation on this mechanism cannot be excluded: (Low to very low) Bax et al. (2019) [42]. |
(iii) a single moving metal mechanism described in [50] | Only heavy atom (non-hydrogen atom) positions defined by experimental data. Mechanism suggests the red hydrogen atom from water 5093 will protonate the 3′ oxygen on the scissile phosphate to cleave the DNA, after which the metaphosphate-like intermediate will be accepted by the catalytic tyrosinate. (High to very high) (Nicholls et al., 2024) [50]. |
(iv) Some unthought-of mechanism | (Low?). No reference. |
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Morgan, H.; Nicholls, R.A.; Warren, A.J.; Ward, S.E.; Evans, G.; Long, F.; Murshudov, G.N.; Duman, R.; Bax, B.D. How Do Gepotidacin and Zoliflodacin Stabilize DNA Cleavage Complexes with Bacterial Type IIA Topoisomerases? 1. Experimental Definition of Metal Binding Sites. Int. J. Mol. Sci. 2024, 25, 11688. https://doi.org/10.3390/ijms252111688
Morgan H, Nicholls RA, Warren AJ, Ward SE, Evans G, Long F, Murshudov GN, Duman R, Bax BD. How Do Gepotidacin and Zoliflodacin Stabilize DNA Cleavage Complexes with Bacterial Type IIA Topoisomerases? 1. Experimental Definition of Metal Binding Sites. International Journal of Molecular Sciences. 2024; 25(21):11688. https://doi.org/10.3390/ijms252111688
Chicago/Turabian StyleMorgan, Harry, Robert A. Nicholls, Anna J. Warren, Simon E. Ward, Gwyndaf Evans, Fei Long, Garib N. Murshudov, Ramona Duman, and Benjamin D. Bax. 2024. "How Do Gepotidacin and Zoliflodacin Stabilize DNA Cleavage Complexes with Bacterial Type IIA Topoisomerases? 1. Experimental Definition of Metal Binding Sites" International Journal of Molecular Sciences 25, no. 21: 11688. https://doi.org/10.3390/ijms252111688