The Structure of the Minimal Relaxase Domain of MobA at 2.1 Å Resolution

Abstract

SUMMARYThe plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 Å X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well-defined target DNA called oriT. The active site metal is observed near Tyr 25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism. KeywordsMobA; X-ray structure; DNA binding; drug resistance; relaxases Resistance to antibiotics by bacterial pathogens is a major health concern throughout the world. Bacteria acquire resistance to antibiotics most commonly by the conjugative transfer of plasmids and transposable elements. There are two classes of plasmids involved in conjugative transfer. One of these consists of large (greater than 30 Kb) self-transmissible elements 1 and the other smaller (~5-10 Kb) mobilizable plasmids 2 3 . Both groups encode the proteins required to process the DNA for transfer, but only the self-transmissible plasmids encode as well the complex, type IV secretion system (T4SS) required to transport the DNA into a new cell. The mobilizable plasmids use a T4SS that is generally provided by a co-resident, selftransmissible plasmid 4 , but can also take advantage of type IV systems involved in the transport of effector proteins active in pathogenesis 5 . An important characteristic of T4SS is that it can transport both protein and DNA across species barriers 6, 7 and even into cells of different kingdoms 8; 9 . In addition, some groups of plasmids are maintained in a large numberCorrespondence to: Jon D. Robertus. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of different bacterial species 10 . These two characteristics have together resulted ...

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…Crystal structures have been presented for DNA strand transferases: (i) a rolling-circle replicase protein from adeno-associated virus (126), (ii) the relaxase domain of TraI from the F plasmid without and with a bound DNA substrate (72,166,167,253), (iii) the relaxase domain of TrwC from plasmid R388 with bound DNA (31,117), and (iv) the relaxase domain of MobA from IncQ plasmid R1162 (193). (Hereafter, we will identify a specific T4SS or subunit by the system or subunit name followed by a subscript indicating the associated conjugative element or organism, e.g., VirB/VirD4 At [for the A. tumefaciens VirB/VirD4 system], TraI F , TrwC R388 , and MobA R1162 ).…”

Folded DNA in Action: Hairpin Formation and Biological Functions in Prokaryotes

“…The structures of two relaxases other than TrwC have been determined by crystallography: the F-plasmid relaxase TraI (35) and the R1162 plasmid relaxase MobA (117). Although they show poor sequence homology to TrwC, the three-dimensional (3D) structures of all these relaxases are very similar.…”

Mobile genetic elements in the genus Bacteroides, and their mechanism(s) of dissemination

“…80 Crystal structures of some relaxases have been obtained, including that of the F plasmid TraI nicking domain, with and without a bound DNA substrate, 86,87 the nicking domain of TrwC from plasmid R388 with bound DNA 88,89 and that of MobA from Inc.Q plasmid R1162. 90 In many cases, the nicking domain itself contains at least three conserved protein motifs. Motif I contains the active site tyrosine, which creates a single-stranded 5' DNA nick through a trans-esterification reaction similar to that of type I topoisomerase.…”