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Comparative Study
. 2000 Nov 1;19(21):5625-34.
doi: 10.1093/emboj/19.21.5625.

The solution structure of the C-terminal domain of the Mu B transposition protein

L H Hung  1 , G Chaconas, G S Shaw
Affiliations
Comparative Study

The solution structure of the C-terminal domain of the Mu B transposition protein

L H Hung et al. EMBO J. .

Abstract

Mu B is one of four proteins required for the strand transfer step of bacteriophage Mu DNA transposition and the only one where no high resolution structural data is available. Structural work on Mu B has been hampered primarily by solubility problems and its tendency to aggregate. We have overcome this problem by determination of the three-dimensional structure of the C-terminal domain of Mu B (B(223-312)) in 1.5 M NaCl using NMR spectroscopic methods. The structure of Mu B(223-312) comprises four helices (backbone r.m.s.d. 0.46 A) arranged in a loosely packed bundle and resembles that of the N-terminal region of the replication helicase, DnaB. This structural motif is likely to be involved in the inter-domainal regulation of ATPase activity for both Mu A and DnaB. The approach described here for structural determination in high salt may be generally applicable for proteins that do not crystallize and that are plagued by solubility problems at low ionic strength.

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Figures

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Fig. 1. 500 MHz 1H-15N HSQC spectrum of uniformly 15N-labeled Mu B223–312 in 20 mM Na2HPO4, 1.5 M NaCl pH 6.8 at 25°C. Residue assignments are labelled according to sequence numbering for the intact protein. Side chain resonances are each connected with a line and labelled in parentheses. The box indicates the position of the E290 crosspeak that is visible at higher contour levels.
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Fig. 2. Solution structures of Mu B223–312 as determined from NMR data. (A) The backbone atoms (N, Cα, CO) for the family of 20 converged structures are shown using the superposition of helices α1 (K235–A245), α2 (E251–Q259), α3 (A266–A278) and α4 (N289–R298). The initial eight residues have no regular structure and were omitted from the figure. (B) Ribbon diagram of the ensemble average structure for the 20 structures in the same orientation as (A). Also shown are the side chains from residues W246 (α1), L275, L277 and A278 (α3) and Y292, L293, A296 and F297 (α4) that comprise the core region of the Mu B223–312 domain. (C) Details of the core region for Mu B223–312 showing the high degree of structural convergence for side chains of all 20 structures.
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Fig. 3. Comparison of the folds of Mu B223–312 and the N-terminal region of E.coli DnaB. (A) The structures are shown side by side and superimposed as ribbon representations. Helices α1 and α2 were used to align the two proteins (backbone r.m.s.d. 1.62 Å). The resulting superposition of the two proteins was performed for residues K235–A278 of Mu B223–312. (B) Interhelical angles and distances between the first three helices of Mu B and DnaB.
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Fig. 4. Multiple sequence alignment of helices α1, α2 and α3 from Mu B223–312 and the N-terminal region for DnaB from different organisms. The structural alignment in Figure 3 was used to align the sequences. Residues that are conserved between Mu B223–312 and DnaB are shaded. Residues that are identical in all DnaB proteins are boxed. Sequence numbering is indicated for Mu B (top) and E.coli DnaB (bottom).
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Fig. 5. Packing similarity for helices α1 and α3 of Mu B223–312 (A) and the N-terminal region region of E.coli DnaB (B). (A) In Mu B223–312 the side chain of W246 from helix α1 inserts itself between the two helices to fortify the packing between A245 and A277. (B) In DnaB, the analogous packing arrangement shows the longer side chains of L40 and M71 at the helix–helix interface. (C) Superposition of helices α2 from Mu B223–312 (blue) and DnaB (gold) are shown using the same backbone alignment as in Figure 3. The side chains that form the negatively charged face are shown. Also indicated are the conserved residues V36, V50 and V54 in DnaB and A241, L256 and I260 in Mu B, which act to maintain the proper orientation of helix α2 with helices α1 and α3.
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Fig. 6. Poisson–Boltzmann charge distribution for Mu B223–312 calculated using GRASP (Nicholls, 1992). (A) The charge distribution was mapped to the Connolly surface of Mu B223–312 with positively charged basic (blue) and negatively charged acidic (red) areas indicated. (B) Same as (A) but rotated ∼120° in a clockwise direction around the x-axis. On the right side of each surface diagram is a ribbon representation showing the side chains of basic residues found at the surface of the protein.
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References

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