The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold

Abstract

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares > or =60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a beta-barrel/sandwich formed by eight antiparallel beta-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site.

Figure 1.

¹H-¹⁵N HSQC spectrum of Atu4866 recorded at 25°C and pH 5.0. (Horizontal lines) Resonances of Asn and Gln side chain protons attached to a common nitrogen, (ε1) tryptophan indole NH proton. Signals originated from residues in the His-tag were not labeled.

Figure 2.

(A) Stereoview of the 20 lowest-energy structure ensemble of Atu4866. The numeric labels denote residue numbers. (B) Ribbon representation of Atu4866 in the same orientation as in A and after a 90-degree rotation about the vertical axis. The secondary structure elements are labeled sequentially from the N to C terminus. (Magenta) β-strands, (salmon) loops. This figure was generated using PyMOL (DeLano Scientific).

Figure 3.

(A) Sequence alignment of Atu4866 and 20 other hypothetical proteins identified by BLAST search. Multiple sequence alignment was performed using Clustal W (Thompson et al. 1994), and the diagram was generated using Jalview (Clamp et al. 2004). “Conservation” is a numerical index calculated by Jalview based on the method proposed by Livingstone and Barton (1993). (Diamonds) Fully conserved (or identical) residues, (+) highly conserved residues with conservation between 9 (light blue shading), and fully conserved (deep blue shading). (B) A ribbon representation of the lowest-energy Atu4866 structure. (Sticks) Side chains of conserved residues, (deep green) highly conserved residues, (lime green) conserved residues. (C) Surface representation of the Atu4866 structure in the same orientation and color scheme as in B. The figure was generated using PyMOL (DeLano Scientific).