Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases.

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Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

Echinomycin is a nonribosomal peptide antibiotic that acts by intercalating double‐stranded DNA. The echinomycin biosynthetic gene cluster of Streptomyces lasaliensis contains a gene of unverified function, ecm16. We show that expression of Ecm16 in the echinomycin‐sensitive E. coli K12 renders cells resistant to echinomycin. Additionally, we have determined the X‐ray crystal structure of Ecm16 at 2.0 Å resolution. Interestingly, the three‐dimensional structure of Ecm16 resembles that of UvrA, the DNA damage sensor protein from the prokaryotic nucleotide excision repair pathway. Ecm16, like UvrA, contains two nucleotide binding domains on a single polypeptide chain, and it can bind double‐stranded DNA. However, our results show that neither UvrA nor Ecm16 can complement each other's function in vivo.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the Nucleotide Excision Repair (NER) protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 preferentially binds double-stranded DNA over single-stranded DNA and is likely to primarily interact with the backbone of DNA using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased significantly when the DNA is intercalated with echinomycin. Ecm16 can repair echinomycin-induced DNA damage independently of NER. Like UvrA, Ecm16 has ATPase activity and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Increasing the cellular levels of UvrA in E. coli was insufficient to render echinomycin resistance. Similarly, Ecm16 was unable to repair DNA damage that is specific to UvrA. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin. Our results, together with past observations, suggest a model where Ecm16 recognizes double helix distortions caused by echinomycin and repairs the problem independently of NER.