Supporting Information

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Table 1. Summary of structure statistics for TraO^{CT I}

*<SA> represents the set of 20 selected conformers obtained by restrained dynamical simulated annealing in CNS/Xplor-NIH. SA_lowest refers to the lowest energy structure of the set.

SI Text

Previous work suggested that the interaction between VirB7 and VirB9 homologues occurs between VirB7 and the C-terminal region of VirB9 (1-4). For this study, we expressed soluble versions of VirB7 that lacked lipidation (see Supporting Methods, below, for details). We were unable to form a complex between Brucella suis VirB9^CT and VirB7 except when we coexpressed the two proteins (SIb Fig. 6B). Unfortunately, this complex did not crystallize. We then focused our attention on the corresponding proteins from pKM101 TraO (TraO^CT residues 177-294) and TraN (residues 17-49). A complex between the two proteins formed after expression and purification of the proteins separately or after coexpression and copurification (SI Fig. 6C Upper). We were also unable to obtain crystals of the pKM101 proteins. We therefore turned to NMR to solve the structure of these complexes. NMR spectroscopy provides a means to investigate the interactions of macromolecules. More specifically, titrations of a potential binding partner with an isotope-labeled target monitored by 2D heteronuclear spectroscopy are commonly used to identify the binding surface. However, to perform ¹H-¹⁵N HSQC NMR experiments in which ¹⁵N-labeled VirB9 is titrated with unlabeled VirB7 requires the ability to express ¹⁵N-labeled VirB9 separately and to form the complex from the purified proteins. Because this was achieved only for the TraO^CT-TraN complex, we determined the NMR structure of this complex.

We observed that when TraO^CT interacts with TraN, residues at the binding interface experience a differing chemical environment, resulting in a chemical shift change (Dd) of the cross-peak in the ¹H-¹⁵N HSQC spectrum. Comparison of the ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled TraO^CT alone or in a 1:1 molar ratio with unlabeled TraN reveals binding of TraN to TraO^CT (SI Fig. 7A). Binding of TraN is characterized by a concomitant decrease in the cross-peak intensity for free TraO^CT, with an increase in the cross-peak intensity for bound TraO^CT, indicating slow exchange on the NMR time scale, and a slow off-rate, suggesting relatively strong binding. The ¹H-¹⁵N HSQC spectrum of ¹⁵N-labeled TraN (36 residues) in complex with unlabeled TraO^CT shows chemical shift dispersion that is clearly consistent with an ordered conformation (SI Fig. 7B).

Samples of either ¹⁵N- or (¹³C,¹⁵N)-labeled TraO^CT/unlabeled TraN or unlabeled TraO^CT/¹⁵N- or (¹³C,¹⁵N)-labeled TraN were prepared, and full resonance assignments were obtained (5). On the basis of the analysis of 3D ¹⁵N- and ¹³C-separated ¹H-NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, we determined the 3D solution structure of the complex between TraO^CT and TraN. A total of 2,403 NOE-derived interproton distance restraints for TraO^CT/TraN, including 235 interprotein TraO^CT-TraN NOEs, were used in the final iterations of the structure calculations for residues 177-271 of TraO^CT and 21-42 of TraN (SI Table 1). Residues 17-20 and 43-48 of TraN and 272-294 of TraO^CT were omitted from the structure calculations because they displayed low ¹H-¹⁵N heteronuclear NOE values (<0), indicating internal flexibility on a fast time scale (data not shown). Omitting a small number of residues at both the N and C termini of TraO^CT, the refined conformer bundles represent well defined models for the TraO^CT domain with rmsd values of 0.42 ±0.06 Å for the backbone and 0.88 ± 0.09 Å for all of the heavy atoms (residues included in the superposition are 180-266; SI Fig. 7C). TraN has a Ca RMSD of 2Å between the 20 members of the ensemble. Two stretches of the polypeptide chain show a Ca RMSD below 1Å, between Pro-23 and Trp-27 and between Thr-30 and Asn-34 (Fig. 2). These stretches form the main interaction site with TraO^CT (Fig. 1).

BsB7 (residues 17-58) and BsB9^CT (residues 165-289) were cloned into the first and second multiple-cloning sites of pRSF-Duet (Novagen), respectively, and coexpressed in Codonplus RPIL E. coli (Stratagene). The complex was purified by using a Ni column (His-Trap), followed by gel filtration.

The sequence encoding TraN lacking its periplasmic localization and lipid-modification signals (residues 17-49) was cloned in pGEXcs and expressed in Codonplus RPIL E. coli (Stratagene) as a TEV (Tobacco Etch Virus) protease-cleavable GST-fusion. The sequence encoding TraO^CT (residues 177-294) was cloned in pET151-TOPO and was expressed in Codonplus RPIL E. coli (Stratagene) as a TEV-cleavable His₆ fusion protein. TraO^CT was purified by using a Ni column (His-Trap), followed by TEV protease cleavage, removal of TEV protease and His tag by a Ni column, and gel filtration. TraO^CT eluted from the gel filtration as two peaks (SI Fig. 6D), which were separately pooled for NMR spectroscopy. A complex between TraO^CT and TraN was purified by mixing E. coli cell lysates expressing the individual proteins and passing them first through a Ni column (His-Trap) and then binding to a GST-Trap column. The complex was eluted from the GST-Trap column by TEV protease cleavage, and TEV protease and the His tag were removed by a second His-Trap column. The complex eluted as a single peak by gel filtration (SI Fig. 6D). For NMR spectroscopy, protein samples were prepared in a buffer containing 10 mM Na/K phosphate, pH 6.7, 100 mM NaCl, 1 mM EDTA, and 0.2% sodium azide. ¹⁵N- or ¹³C-labeled proteins were produced by growing E. coli in minimal media supplemented with ¹⁵N ammonium sulfate (Sigma) or ¹³C D-glucose (Sigma).

Sequence-specific resonance assignment of TraO^CT in the absence of TraN was obtained by using standard triple resonance experiments, namely: HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HA(CA)NH, and HA(CACO)NH. By using the ¹³Ca, ¹³Cb, ¹³CO, and ¹Ha chemical shift values, TraO^CT in the absence of TraN is predicted by the Chemical Shift Index method (10) to have the same secondary structure elements as TraO^CT in complex with TraN.

All structures for TraO^CT/TraN were calculated by using an ab initio simulated annealing protocol within the CNS program (12), with PARALLHDGv5.3 force field and PROLSQ nonbonded energy function (13). The protocol adopts a mixture of Cartesian molecular dynamics (MD) and torsion angle dynamics-simulated annealing to refine structures starting from random-generated conformers with good local geometry.

A total of 2403 NOE-derived interproton distance restraints for TraO^CT/TraN were included in the final iterations of the structure calculations (see SI Table 1). Backbone torsion angle restraints for j and y were derived from analysis of ¹Ha, ¹³Ca, ¹³Cb, ¹³C', and ¹⁵N^H chemical-shift databases as implemented in the program TALOS (14). Hydrogen-bond restraints for amide protons were derived from an assessment of the regular secondary structure elements. A total of 118 dihedral angle and 54 hydrogen bond (27 H-bonds; 2 distance restraints per H bond) interatomic distance restraints were used for TraO^CT/TraN.

The atomic coordinates of the final 20 simulated annealing TraO^CT/TraN conformers and the list of experimental restraints (PDB ID code 2OFQ) has been deposited at the RCSB Protein Data Bank. Chemical shifts for resonance assignments for the TraO^CT/TraN complex have been deposited at the BioMagResBank (accession code 6936).

Cys substitution mutations in VirB9 were generated by QuikChange (Stratagene) or the Kunkel method (20) using pJW373 as a template. For pBALM1 producing VirB9N216C, the mutagenic oligonucleotides were VirB9N216Cs 5'-CTTTCCCGGGTGCGTACGCATACCC-3' and VirB9N216Cas 5'-GGGTATGCGTACGCACCCGGGAAAG-3'. For pBALM2 producing VirB9N226C, the mutagenic oligonucleotides were VirB9N226Cs 5'-CTACACCATCTGCCCTGACGGCAAGGAAGC-3' and VirB9N226Cas 5'-GCTTCCTTGCCGTCAGGGCAGATGGTGTAG-3'. For pBALM4 producing VirB9D228C, the mutagenic oligonucleotides were VirB9D228Cs 5'-CATCAATCCTTGCGGCAAGGAAGCTGTC-3' and VirB9D228Cas 5'-GACAGCTTCCTTGCCGCAAGGATTGATG-3'. For pBALM3 producing VirB9P227C, ssDNA prepared from pJW373 served as the template together with the mutagenic oligonucleotide VirB9227Cas 5'-GCTTCCTTGCCGTCGCAATTGATGGTG-3' according to Kunkel (20)

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