Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion - PubMed (original) (raw)

Comparative Study

. 2005 Feb 1;102(5):1685-90.

doi: 10.1073/pnas.0409399102. Epub 2005 Jan 24.

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Comparative Study

Predicted hexameric structure of the Agrobacterium VirB4 C terminus suggests VirB4 acts as a docking site during type IV secretion

Rebecca Middleton et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The Agrobacterium T-DNA transporter belongs to a growing class of evolutionarily conserved transporters, called type IV secretion systems (T4SSs). VirB4, 789 aa, is the largest T4SS component, providing a rich source of possible structural domains. Here, we use a variety of bioinformatics methods to predict that the C-terminal domain of VirB4 (including the Walker A and B nucleotide-binding motifs) is related by divergent evolution to the cytoplasmic domain of TrwB, the coupling protein required for conjugative transfer of plasmid R388 from Escherichia coli. This prediction is supported by detailed sequence and structure analyses showing conservation of functionally and structurally important residues between VirB4 and TrwB. The availability of a solved crystal structure for TrwB enables the construction of a comparative model for VirB4 and the prediction that, like TrwB, VirB4 forms a hexamer. These results lead to a model in which VirB4 acts as a docking site at the entrance of the T4SS channel and acts in concert with VirD4 and VirB11 to transport substrates (T-strand linked to VirD2 or proteins such as VirE2, VirE3, or VirF) through the T4SS.

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Figures

Fig. 4.

Fig. 4.

MSA of VirB4 and TrwB and selected homologs along their NTP-binding domains. Sequence homologs to VirB4 are shown directly below VirB4, followed by TrwB and homologs. Walker A residues are boxed in red, Walker B residues are boxed in magenta, and the conserved glutamine is highlighted in green.

Fig. 2.

Fig. 2.

VirB4-TrwB pairwise alignment. The cytoplasmic domain of TrwB (residues 122–507) and the C terminus of VirB4 (residues 425–789) are aligned, with identical and similar residues shown in black and gray, respectively. Boxed areas highlight the Walker A and B motifs. The Walker A motif is conserved across VirB4 and TrwB (VirB4, G433-T440; TrwB, G130-S137). The Walker B motif is found in its entirety in VirB4 (R619-E635), whereas a truncated version is evident in TrwB (R349-E357).

Fig. 3.

Fig. 3.

Homology model for the C terminus of VirB4. (A) VirB4 monomer model. α-helices are blue and β-sheets are gold. The Walker A motif (G433-T440) is red, the Walker B motif (R619-E635) is magenta, and the conserved Q668 is green. (B) Monomer of TrwB, using the same coloring as for VirB4. (C) Superposition of VIRB4 (turquoise) and TrwB (gold). (D) Predicted hexameric structure of VirB4.

Fig. 1.

Fig. 1.

Domain organization of VirB4 and TrwB. VirB4 and TrwB share a C-terminal NTP-binding domain but have different N termini. The N terminus of VirB4 matches the

pfam

domain CagE_TrbE_VirB, but has no known function or structure, whereas the TrwB N terminus contains two predicted TM helices.

Fig. 5.

Fig. 5.

Surface image of VirB4 monomer, highlighting the NTP-binding cleft. To better display the NTP-binding cleft, we show the structure from a different perspective, corresponding to the back side of Fig. 3_A_. The conserved lysine (K) of the Walker A motif is red, the conserved DE of the Walker B motif is purple, and the conserved glutamine (Q) is highlighted in yellow.

Fig. 6.

Fig. 6.

Model for the function of VirB4 during the type IV secretion process. VirB4 is shaded in gray as two domains linked by a hinge (solid line). VirB11 and VirD4 are black and stippled, respectively. For simplicity, only two subunits of VirB4, VirD4, and VirB11 are shown, and the rest of the T4SS is drawn as a half cylinder. VirD2 is shown as a white square attached to the T-strand (wave ladder-like line). Arrows indicate subunit exchange between VirB4 and VirD4. See text for details. OM, outer membrane; IM, inner membrane.

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