Biology subjects

Bax, B. D.

Publications and source records attributed to Bax, B. D..

5 recordsLinked to original sources

bioRxiv2024

How do gepotidacin and zoliflodacin stabilize DNA-cleavage complexes with bacterial type IIA topoisomerases? 1. Experimental definition of metal binding sites

One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA-strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1[A] GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a=b{approx}93[A], c{approx}412[A]). This suggests it may be possible to observe the two DNA-cleavage steps (and two DNA-religation steps) in this P61 space-group. Here a 2.58[A] anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98[A], 2.1[A], 2.5[A] and 2.65[A]) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism - presented in an accompanying (2nd) paper. A previously published 2.98[A] structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98[A] yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites.

biophysics↗

bioRxiv2024

How do gepotidacin and zolifodacin stabilize DNA-cleavage complexes with bacterial type IIA topoisomerases? 2. A Single Moving Metal Mechanism.

DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water-metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA-breaks. Gepotidacin had been observed to sit on the twofold axis of the complex, midway between the two four base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA-breaks. Here we use information from three crystal structures of complexes of Staphlococcus aureus DNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA-cleavage. Movement of a single catalytic metal-ion (Mg2+ or Mn2+) guides water mediated protonation and cleavage of the scissile phosphate which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally how such mechanisms can be experimentally distinguished is considered.

biophysics↗

bioRxiv2022

A 2.8A structure of zoliflodacin in a DNA-cleavage complex with Staphylococcus aureus DNA gyrase

Since 2000 some thirteen quinolones/fluoroquinolones have been developed and come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA-cleavage complexes with DNA gyrase and topo IV, the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here we report a 2.8 [A] X-ray crystal structure which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA-cleavage site(s) as quinolones sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA) suggesting it may be more difficult for bacteria to develop target mediated resistance. We found that zoliflodacin had an MIC of 4 {micro}g/mL against Acinetobacter baumannii, an improvement of 4-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, are likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

biophysics↗

bioRxiv2021

Structures of the Human SPAK and OSR1 Conserved C-Terminal (CCT) Domains

STE20/SPS1-related proline/alanine-rich kinase (SPAK) and Oxidative Stress Responsive 1 (OSR1) kinase are two serine/threonine protein kinase that regulate the function of ion co-transporters through phosphorylation. The highly conserved C-terminal (CCT) domains of SPAK and OSR1 bind to RFx[V/I] peptide sequences from their upstream With No Lysine Kinases (WNKs), facilitating their activation via phosphorylation. Thus, the inhibition of SPAK and OSR1 binding, via their CCT domains, to WNK kinases is a plausible strategy for inhibiting SPAK and OSR1 kinases. To facilitate structure-guided drug design of such inhibitors, we expressed and purified human SPAK and OSR1 CCT domains and solved their crystal structures. We also employed a biophysical strategy and determined the affinity of SPAK and OSR1 CCT domains to an 18-mer peptide derived from WNK4. Together, the crystal structures and affinity data reported herein provide a robust platform to facilitate the design of CCT domain specific small molecule inhibitors of SPAK-activation by WNK kinases, potentially leading to new improved treatments for hypertension and ischemic stroke.

biochemistry↗

bioRxiv2021

Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a closed hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the ATP a-phosphate. In contrast, in open hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzymefragment structures show that Tyr121 is flipped out of its pocket, suggesting that this pocket is druggable.

biophysics↗