Structural analysis of Brucella abortus RicA substitutions that do not impair interaction with human Rab2 GTPase
© Nkengfac et al.; licensee BioMed Central Ltd. 2012
Received: 24 July 2012
Accepted: 30 July 2012
Published: 14 August 2012
Protein-protein interactions are at the basis of many cellular processes, and they are also involved in the interaction between pathogens and their host(s). Many intracellular pathogenic bacteria translocate proteins called effectors into the cytoplasm of the infected host cell, and these effectors can interact with one or several host protein(s). An effector named RicA was recently reported in Brucella abortus to specifically interact with human Rab2 and to affect intracellular trafficking of this pathogen.
In order to identify regions of the RicA protein involved in the interaction with Rab2, RicA was subjected to extensive random mutagenesis using error prone polymerase chain reaction. The resulting allele library was selected by the yeast two-hybrid assay for Rab2-interacting clones that were isolated and sequenced, following the “absence of interference” approach. A tridimensional model of RicA structure was used to position the substitutions that did not affect RicA-Rab2 interaction, giving a “negative image” of the putative interaction region. Since RicA is a bacterial conserved protein, RicA homologs were also tested against Rab2 in a yeast two-hybrid assay, and the C. crescentus homolog of RicA was found to interact with human Rab2. Analysis of the RicA structural model suggested that regions involved in the folding of the “beta helix” or an exposed loop with the IGFP sequence could also be involved in the interaction with Rab2. Extensive mutagenesis of the IGFP loop suggested that loss of interaction with Rab2 was correlated with insolubility of the mutated RicA, showing that “absence of interference” approach also generates surfaces that could be necessary for folding.
Extensive analysis of substitutions in RicA unveiled two structural elements on the surface of RicA, the most exposed β-sheet and the IGFP loop, which could be involved in the interaction with Rab2 and protein folding. Our analysis of mutants in the IGFP loop suggests that, at least for some mono-domain proteins such as RicA, protein interaction analysis using allele libraries could be complicated by the dual effect of many substitutions affecting both folding and protein-protein interaction.
KeywordsProtein-protein interaction Yeast two-hybrid Mutagenesis Brucella
Brucella abortus is a facultative intracellular pathogen responsible for a worldwide zoonosis . Like other intracellular bacteria such as Legionella spp [2–4] and Salmonella spp  , B. abortus probably depends on precisely orchestrated interactions with host cell proteins for its infectious process. Remarkably, these intracellular pathogens secrete proteins regulating host small GTPases [4–7]. Small GTPases of the Ras super family are signaling proteins that cycle between a GDP-bound inactive state and a GTP-bound active state. These two states are regulated by guanine-nucleotide exchange factors, which facilitate the conversion of GDP to GTP; GTPase activating proteins, which facilitate the hydrolysis of the GTP and Guanine-nucleotide-dissociation inhibitors, which negatively regulate the exchange activity of the GTPase and dislocate them from membranes. Rab GTPases are small GTPases playing a critical role in the control of membrane trafficking. Specifically, Rab2 has been shown to control membrane trafficking between the Golgi apparatus and the endoplasmic reticulum , Rab2 was also putatively associated with the phagosome  but without any known function in phagosomal maturation in mammalian cells . RicA is an effector recently identified in B. abortus, which interacts with human Rab2 . This interaction was detected using yeast 2-hybrid (Y2H) and confirmed by GST-pulldown. RicA has a preference for GDP-bound GST-Rab2 compared to GTPγS-bound GST-Rab2 . Active Rab2 is known to be required for B. abortus intracellular proliferation . A B. abortus ΔricA strain recruits less Rab2 on the Brucella containing vacuole, suggesting that RicA is playing a role during the intracellular trafficking of the bacterium .
RicA is predicted to belong to the superfamily of LβH proteins, comprising acetyltransferases, acyltransferases, carbonic anhydrases, ferripyochelin binding proteins, as well as many proteins of unknown functions. Their structure is characterized by the assembly of three β sheets in a left-handed “β helix” structure. In this paper, we attempted to localize the Rab2 interaction surface on the RicA predicted structure. We performed the “absence of interference” approach  previously used to map the interface of the catalytic domain of the DNA methylase Dnmt3a and its regulatory factor Dnmt3L. Mapping of the substitutions that do not disrupt the RicA-Rab2 interaction, on the predicted model of RicA structure, revealed two possible interfaces, a beta sheet and a loop called IGFP. The data reported here suggested that, of these two structural elements, at least the IGFP loop is also involved in RicA folding.
Prediction of RicA three-dimensional structure
RicA mutagenesis and selection of alleles allowing interaction with Rab2
Distribution of substitutions in the mutated RicA clones interacting with Rab2 in a Y2H assay
Substitutions (and their score in the Blosum62 matrix)
G8E (−2), G38S (0), D148V (−3)
N132Y (−2), R144C (−3)
P2S (−1), D24H (−1), M101T (−1), V135A (0)
M127T (−1), E163D (2)
I99N (−3), H166R (0)
P2S (−1), D24H (−1), I76N (−3), M101T (−1), V135A (0)
K10M (−1), P51T (−1)
E17G (−2), K124E (1)
I21V (3), E128V (−2), L143Q (−2)
L45M (2), A119V (0)
K10E (1), M172L (2)
T92S (1), A150T (0), G171C (−3)
Rab2 interaction assay with RicA homologs
Since B. abortus RicA is conserved in many other bacteria, we tested the interaction of RicA homologs with human Rab2, using Y2H. Interestingly, by fusing Caulobacter crescentus RicA homolog to the AD of Gal4p, we detected interaction with human Rab2 in a Y2H assay using the HIS3 and URA3 reporters. The C. crescentus RicA homolog is sharing 52% identities with B. abortus RicA, indicating that it has a similar fold but with many substitutions, strongly suggesting that only conserved residues contribute to the interaction between RicA and Rab2.
Structural analysis of the substitutions that do not impair RicA-Rab2 interaction
Mutagenesis of the IGFP loop
The IGFP loop is part of a sequence of 8 amino acids in length flanked on each side by a β strand of the LβH (Figure 1). The sequence of four amino acids Ile-Gly-Phe-Pro (IGFP) is exposed to the surface of RicA in the 3D model of the trimer (Figures 1 and 2). Loop regions of the LβH-containing proteins are known to contribute most of the residues that interact with binding partners [18, 19] and surface hydrophobicity has been used to identify regions of a protein surface most likely to interact with a binding ligand . We therefore proposed that this exposed IGFP loop could be involved in the recognition of Rab2.
Sequenced IGFP replacements that disrupted (clones 1 to 12) or not (clones 13 to 15) RicA-Rab2 interaction
Y2H assay with Rab2
Y GG P
W GV P
T GV P
R GG P
A GH P
V GN P
L GE P
S G* P
A GS P
L GW P
L GH P
A GY P
S GF P
V GF P
K GL P
In order to test a possible alteration of RicA-Rab2 interaction using GST pulldown, we attempted to overproduce XGXP clones n°2, 3, 4, 5, 6 and 8 as His6-RicA fusions. Among the 6 clones tested, none were found in the soluble extract and all were detected in the insoluble pellet, while the wild type control was found exclusively in the soluble fraction. This observation strongly suggests that mutations in the IGFP loop contribute to the proper folding of His6-RicA, at least in E. coli.
The objective of this study was to experimentally identify and characterize protein-protein interaction site of RicA for Rab2, to provide a better understanding of the structural basis of a human small GTPase recognition by a bacterial effector protein. In the 3D model of RicA, the protein may be divided in two parts: a N-terminal LβH component and a C-terminal α helix. Within the LβH, there are three β sheets, two embedded in the trimeric structure and one exposed to the exterior. The two internal β sheets are predicted to form the interfaces between monomers and are rarely substituted (Figure 3) in mutants generated and still able to interact with Rab2, or in the C. crescentus homolog. However, the loops involved in the formation of the central pore of the RicA model (visible in Figure 2B and 2C) are often mutated. The residues of the C-terminal α helix in contact with the LβH component are rarely mutated, while many exposed residues of this α helix are substituted in the mutated RicA or in the C. crescentus homolog. These data are consistent with the proposed 3D model of RicA.
Our data suggest that mutations in the IGFP loop that impair interaction with Rab2 also generate a folding problem. This is rather surprising because the IGFP loop is not very well conserved (except for the G and P residues, see Additional file 4: Figure S1), and moreover it is exposed to the surface of the homologous proteins of known structure ( Additional file 4: Figure S2). The role of the IGFP loop is unknown but it seems to be needed for the generation of a correct tertiary or quaternary structure, since the 6 XGXP mutants unable to interact with Rab2 are found to be insoluble when expressed in E. coli, while the wild type RicA is soluble. This data indicates that regions necessary for folding could overlap the regions necessary for interaction between RicA and Rab2, unless the LS substitutions do not affect interaction between RicA and Rab2. Indeed, we cannot exclude that the RicA-Rab2 interaction is sufficiently stable to be resistant to point mutations, which would preclude most of the strategies targeting loss-of-interaction mutants.
In conclusion, we predict that for a fraction of mono-domain proteins, including B. abortus RicA, some structural element(s) like the IGFP loop could be bifunctional, involved in both protein folding and protein-protein interaction, as depicted in Figure 4.
B. abortus 2308 ricA coding sequence was cloned in the Gateway entry vector pDONR201 and sequenced before sub-cloning in pAD vector as a fusion protein with Gal4 activation domain (AD) and served as the prey plasmid (pAD-RicA). Human Rab2 coding sequence was cloned in pDB vector as a fusion protein with Gal4 DNA binding domain (BD) and used as the bait (pDB-Rab2). The ricA coding sequence was also cloned in pET15b vector (Novagen pET expression system, pET15b-RicA) as a fusion protein with N-terminal hexahistidine tag (His6-RicA). The ricA coding sequence was PCR amplified with primers that introduced N-terminal Nde I site and a C-terminal Bam HI site (Nde I RicA-F: 5’CAT ATG CCG ATC TAT AAC GG; Bam HI RicA-R: 5’GGA TCC TCA GGC AGG CTC CAT). The pET15b-RicA construct was checked by restriction diagnosis and sequencing (primer, T7: 5’TAA TAC GAC TCA CTA TAG GG).
Generation of random mutagenesis, site directed mutagenesis and sequencing
The ricA mutagenesis fragment (525 bp) was synthesized by error-prone PCR  on the expression clone pAD-RicA with primers that hydridize to att B1 and att B2 sites that flank ricA in the pAD-RicA (att B1-F 5′ACA AGT TTG TAC AAA AAA GCA G-3′; att B2-R 5’ AC CAC TTT GTA CAA GAA AGC T-3′). Following PCR, DNA was purified and cloned into pDONR201 and the att L1 site primer (5′-CTGAAGCTTGGATCTCGGGC-3′) was used for sequencing. The generated random mutant library (entry clones) was sub-cloned into pAD expression vector.
Site directed mutations were incorporated into the IGFP motif using the MutagenexTM Library method. The residues Ile-70 and Phe72 were replaced with random amino acids (amino acid switches from IGFP to XGXP, where X may be one of the 20 possible amino acids). The pAD-RicA plasmid was used as template for a PCR with four synthetic oligonucleotide primers, two containing the desired mutations (FM 5′ ATG CAC ACC GAT NNK GGC NNK CCG CTG ACC ATC 3′; RM 5′ GAT GGT CAG CGG MNN GCC MNN ATC GGT GTG CAT 3′) (where N is any of A, C, G, or T; K is G or T; M is A or C) which are complementary to opposite strands of the insert, and two hybridizing to att B1 and att B2. PCR amplifications were performed for upstream and downstream regions of the mutations. A third assembly PCR was performed with att B1 and att B2 primers, using upstream and downstream PCR fragments as initial substrates. The final PCR products were cloned into pDONR201 to generate a large pool of entry-clones. We sequenced 5 randomly picked clones using the att L1 site primer to confirm the expected site directed mutagenesis of Ile-70 and Phe-72 codons. The entry-clone plasmidic DNA library was prepared and sub-cloned into pAD destination vector. DNA sequencing experiment was performed with the “Standard Sequencing Run" on an ABI PRISM® 3100 Genetic Analyser (Applied Biosystems). Y2H-AD (5′-CGC GTT TGG AAT CAC TAC AGG G 3′ and Y2H-Term (5′-GGA GAC TTG ACC AAA CCT CTG GCG 3′) primers were used to sequence RicA mutants still interacting with Rab2 in the Y2H.
RicA or RicA allele libraries and Rab2 were transformed into MaV203 yeast strain. MaV203 contains single copies of each three reporter genes (HIS 3, URA 3 and lac Z) that are stably integrated at different loci in the yeast genome. The interaction between RicA or its allele and Rab2 reconstituted an active transcription factor, hence the expression of reporter genes. HIS 3 gene expression was detected by plating transformants on selective medium lacking leucine, trytophan and histidine in the presence of 3AT (20 mM). The lac Z reporter was tested by β-galactosidase filter assay. All controls were carried out with appropriate co-transformed vectors.
Mapping and display of mutations on the surface of the protein
The mutations were mapped on the proposed RicA structure using MacPymol (http://www.pymol.org/). The proposed structure was obtained using EsyPred3D program  and verified for correctness using verify3D  server (http://nihserver.mbi.ucla.edu/Verify_3D/).
Expression and purification of His6-RicA fusion
Purified His6-RicA was obtained from E. coli BL21 (DE3) over-expression clone. A filtered lysate was loaded into a chromatography column (Econo-Pac® cat. n° 732–1010, Biorad) pre-loaded with Ni-NTA His-Bind superflow (cat n° 70691–3, Novagen®) resin followed by wash steps and elution (45 mM Tris–HCl pH 7.9, 500 mM NaCl, 200 mM imidazole pH 7.9 and 10% glycerol).
Gal4p transactivation domain
Gal4p DNA binding domain
This work was supported by FRFC (Fonds de la Recherche Fondamentale Collective, conventions n°2.4521.04 and 2.4541.08) from FRS-FNRS (Fonds de la Recherche Scientifique – Fonds National de la Recherche Scientifique) as well as ARC programs (Actions de Recherches Concertées, conventions 04/09-325 and 08/13-015, French Speaking Community of Belgium). Bernard Nkengfac was recipient of a PhD fellowship from the University of Namur.
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