Structural studies on Mycobacterium tuberculosis RecA: Molecular plasticity and interspecies variability

Abstract

Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the 'switch' residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. The interspecies variability in the plasticity of the two mycobacterial proteins is particularly surprising as they have similar sequence and 3D structure. Details of the interactions of ligands with the protein, characterized in the structures reported here, could be useful for design of inhibitors against M. tuberculosis RecA.

Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

“…Despite the fact that Mg 2+ ion was present in the crystallization solution there was no electron density for this ion around the nucleotide ADP to suggest proper coordination. Mg 2+ ion was also not observed in Deinococcus radiodurans and Mycobacterium tuberculosis RecA crystal structures [ 53 , 58 ]. We observed an extra electron density beyond the β-phosphate of ADP which could not be properly modeled with waters, SO 4 3- or PO 4 3- ions.…”

“…The crystallography 6 1 symmetry leads to the formation of a helical filament that has a pitch of 91.3 Å ( Fig 9 ), a value close to the pitch range 90–100 Å determined by electron microscopy for active filaments of EcRecA formed in the presence of DNA, ATP-γ-S or ATP [ 70 ]. Inactive and compressed filaments characterized to date have a helical pitch of 65–85 Å and are formed by RecA alone or bound to ADP, either in the absence of DNA or bound to ssDNA or dsDNA [ 10 , 26 , 51 , 53 , 58 , 71 ]. Despite the fact that HsRecA-ADP/ATP protein was crystallized with ADP and ATP, our structure presented a helical pitch characteristic of an active RecA filament form.…”

“…The monomeric structure of native HsRecA protein exhibits an architecture similar to that of bacterial RecA proteins previously crystallized. HsRecA protein has a small NTD, a core ATPase domain and a large CTD, with the same secondary structure elements of bacterial RecA proteins [ 9 , 51 , 53 , 58 ]. However, the crystallography 6 1 symmetry leads to the formation of a helical filament that has a pitch of 91.3 Å ( Fig 9 ), a value within the pitch range of 90–100 Å determined by electron microscopy for active filaments of EcRecA formed in the presence of DNA, ATP-γ-S or ATP [ 70 ].…”

Structure of F 1 -ATPase from the obligate anaerobe Fusobacterium nucleatum

“…Citrate was present in the crystallization buffer, and it fits the density in molecule 1 better than in molecule 2 (electronic supplementary material, figure S6E,F). It has been found to be bound to the P-loop of RecA from M. smegmatis [57]. Although the crystallization buffer contained 500 mM ADP, similar to the conditions used with the F 1 -ATPase from C. thermarum (5ik2) [52] and M. smegmatis (6foc) [51], no ADP was added to the buffer for harvesting the crystals of the F. nucleatum F 1 -ATPase, and its absence probably accounts for the low occupancy in this site compared to the C. thermarum and M. smegmatis enzymes.…”

Some general aspects of ion interactions with the channel pore

“…In doing so, one obtains the generalized Ramachandran attraction formula (see eqn (6)). 8,19,34,35 Here, eqn (7) is the unique and generalized Ramachandran formula such that it reduces to the standard vdW formula when the interaction between two atoms is much smaller. Eqn (7) is also applicable for intermolecular interaction between other non-bonding polarizable atoms (including hydrogen, halogen and carbon bonds).…”