Tetratricopeptide Repeat Motifs Research Articles

Comparative functional genomics of the TPR gene family in Arabidopsis, rice and maize

The tetratricopeptide repeat (TPR) proteins found in all kingdoms of life can mediate protein-protein interactions in a variety of biological systems through binding to specific peptide ligands, such as cell cycle control, transcription, protein transport and protein folding. Although the proteins are ubiquitous, no comprehensive overview of them in Arabidopsis thaliana (At), Oryza sativa (Os), Zea mays (Zm) and Populus trichocarpa (Pt) has been available in the literature till now. Through whole genome investigation, 177 Arabidopsis, 216 rice, 211 maize and 243 poplar TPR genes were identified and categorized into 28 subfamilies and 11 groups based on their domain compositions and phylogenetic relationships, respectively. Phylogenetic analysis revealed that most genes in the same subfamilies are classified into at least two groups, implying that TPR proteins have acquired functional diversity by extensive domain shuffling and/or emerged multiple times independently during evolution. Structural analysis of ZmTPR080 showed that eight TPR motifs as two anti-parallel α-helices form an amphipathic groove capable of accepting a target protein peptide. Similar expression patterns across development stages suggested functional conservation between homologous pairs, for example, AtTPR050/ZmTPR049 may have similar functions in the regulation of flowering. Under drought stress, 26 OsTPRs showed strong alterations of their expression levels in rice leaf, while 10 and 49 ZmTPRs were significantly differentially expressed in maize leaf and cob, respectively.

Tetratricopeptide repeat domain 3 overexpression tends to form aggregates and inhibit ubiquitination and degradation of DNA polymerase γ.

Tetratricopeptide repeat (TPR) domain 3 (TTC3) is a protein that contains canonical RING finger and TPR motifs. It is encoded by the TTC3 gene located in the Down syndrome critical region (DSCR). In this study, we used a yeast two-hybrid assay to identify several proteins that physically interact with TTC3, including heat shock proteins and DNA polymerase γ (POLG). When TTC3 was overexpressed in mammalian cells, the ubiquitination of POLG was inhibited and its degradation slowed. High TTC3 protein expression led to the development of intracellular TTC3 aggregates, which also contained POLG. Co-expression with Hsp70 or placing the TTC3 gene under control of an inducible promoter alleviated the aggregation and facilitated POLG degradation. As a result of POLG’s effects on aging processes, we propose that a copy number variant of the TTC3 may contribute to Down syndrome pathogenesis.

Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo.

Alcadein α(Alcα) is a major cargo of kinesin-1 that is subjected to anterograde transport in neuronal axons. Two tryptophan- and aspartic acid-containing (WD) motifs located in its cytoplasmic domain directly bind the tetratricopeptide repeat (TPR) motifs of the kinesin light chain (KLC), which activate kinesin-1 and recruit kinesin-1 to Alcα cargo. We found that phosphorylation of three serine residues in the acidic region located between the two WD motifs is required for interaction with KLC. Phosphorylation of these serine residues may alter the disordered structure of the acidic region to induce direct association with KLC. Replacement of these serines with Ala results in a mutant that is unable to bind kinesin-1, which impairs exit of Alcα cargo from the Golgi. Despite this deficiency, the compromised Alcα mutant was still transported, albeit improperly by vesicles following missorting of the Alcα mutant with amyloid β-protein precursor (APP) cargo. This suggests that APP partially compensates for defective Alcα in anterograde transport by providing an alternative cargo receptor for kinesin-1.

The TPR domain of BepA is required for productive interaction with substrate proteins and the β-barrel assembly machinery complex.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β-barrel assembly machinery (BAM) driving integration of β-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.

TaFlo2-A1, an ortholog of rice Flo2, is associated with thousand grain weight in bread wheat (Triticum aestivum L.)

BackgroundThe Flo2 gene is a member of a conserved gene family in plants. This gene has been found to be related to thousand grain weight (TGW) in rice. Its orthologs in hexaploid wheat were cloned, and the haplotype variation in TaFlo2-A1 was tested for association with TGW.ResultsThe cloned sequences of TaFlo2-A1, TaFlo2-B1 and TaFlo2-D1 contained 23, 23 and 24 exons, respectively. The deduced proteins of TaFlo2-A1 (1734 aa), TaFlo2-B1 (1698 aa) and TaFlo2-D1 (1682 aa) were highly similar (>94%) and exhibited >77% similarity with the rice FLO2 protein. Like the rice FLO2 protein, four tetratricopeptide repeat (TPR) motifs were observed in the deduced TaFLO2 protein. An 8-bp InDel (−10 to −17 bp) in the promoter region and five SNPs in first intron of TaFlo2-A1 together formed two haplotypes, TaFlo2-A1a and TaFlo2-A1b, in bread wheat. TaFlo2 was located on homeologous group 2 chromosomes. TaFlo2-A1 was inferred to be located on deletion bin ‘2AL1–0.85-1.00’. The TaFlo2-A1 haplotypes were characterized in the Chinese Micro Core Collection (MCC) and Pakistani wheat collection using the molecular marker TaFlo2-Indel8. TaFlo2-A1 was found to be associated with TGW but not with grain number per spike (GpS) in both the MCC and Pakistani wheat collections. The frequency of TaFlo2-A1b (positive haplotype) was low in commercial wheat cultivars; thus this haplotype can be selected to improve grain weight without negatively affecting GpS. The expression level of TaFlo2-A1 in developing grains at 5 DAF (days after flowering) was positively correlated with TGW in cultivars carrying the positive haplotype.ConclusionThis study will likely lead to additional investigations to understand the regulatory mechanism of the Flo2 gene in hexaploid wheat. Furthermore, the newly developed molecular marker ‘TaFlo2-InDel8’ could be incorporated into the kit of wheat breeders for use in marker-assisted selection.

Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C

Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a β-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24–Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra α-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted α-helix. Such structural feature indicates that the inserted α-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24–Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the α-helical hinge may play important role for exporting glucan chains.

Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

Insights into Dynamic Polymicrobial Synergy Revealed by Time-Coursed RNA-Seq

Many bacterial infections involve polymicrobial communities in which constituent organisms are synergistically pathogenic. Periodontitis, a commonly occurring chronic inflammatory disorder, is induced by multispecies bacterial communities. The periodontal keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii exhibit polymicrobial synergy in animal models of disease. Mechanisms of co-adhesion and community formation by P. gingivalis and S. gordonii are well-established; however, little is known regarding the basis for increased pathogenicity. In this study we used time-coursed RNA-Seq to comprehensively and quantitatively examine the dynamic transcriptional landscape of P. gingivalis in a model consortium with S. gordonii. Genes encoding a number of potential virulence determinants had higher relative mRNA levels in the context of dual species model communities than P. gingivalis alone, including adhesins, the Type IX secretion apparatus, and tetratricopeptide repeat (TPR) motif proteins. In contrast, genes encoding conjugation systems and many of the stress responses showed lower levels of expression in P. gingivalis. A notable exception to reduced abundance of stress response transcripts was the genes encoding components of the oxidative stress-related OxyR regulon, indicating an adaptation of P. gingivalis to detoxify peroxide produced by the streptococcus. Collectively, the results are consistent with evolutionary adaptation of P. gingivalis to a polymicrobial oral environment, one outcome of which is increased pathogenic potential.

The expression patterns of Tetratricopeptide repeat domain 36 (Ttc36)

Tetratricopeptide repeat domain 36 (Ttc36), whose coding protein belongs to tetratricopeptide repeat (TPR) motif family, has not been studied extensively. We for the first time showed that Ttc36 is evolutionarily conserved across mammals by bioinformatics. Rabbit anti-mouse Ttc36 polyclonal antibody was generated by injecting synthetic full-length peptides through “antigen intersection” strategy. Subsequently, we characterized Ttc36 expression profile in mouse, showing its expression in liver and kidney both from embryonic day 15.5 (E15.5) until adult, as well as in testis. Immunofluorescence staining showed that Ttc36 is diffusely expressed in liver, however, specifically in kidney cortex. Thus, we further compare Ttc36 with proximal tubules (PT) marker Lotus Tetragonolobus Lectin (LTL) and distal tubules (DT) marker Calbindin-D28k respectively by double immunofluorescence staining. Results showed the co-localization of Ttc36 with LTL rather than Calbindin-D28k. Collectively, on the basis of the expression pattern, Ttc36 is specifically expressed in proximal distal tubules.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. FimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Interferon Regulatory Factor 8 (IRF8) Impairs Induction of Interferon Induced with Tetratricopeptide Repeat Motif (IFIT) Gene Family Members

The chromosomally clustered interferon-induced with tetratricopeptide repeat motif (IFIT) gene family members share structural features at the gene and protein levels. Despite these similarities, different IFIT genes have distinct inducer- and cell type-specific induction patterns. Here, we investigated the mechanism for the observed differential induction of the mouse Ifit1, Ifit2, and Ifit3 genes in B cells and demonstrated that the repressive effect of the transcription factor interferon regulatory factor 8 (IRF8), which is highly expressed in B cells, played an essential role in this regulation. Although IRF8 could impair induction of all three IFIT genes following stimulation of retinoic acid-inducible gene I (RIG-I), it could selectively impair the induction of the Ifit1 gene following IFN stimulation. The above properties could be imparted to IRF8-non-expressing cells by ectopic expression of the protein. Induction of reporter genes, driven by truncated Ifit1 promoters, identified the regions that mediate the repression, and a chromatin immunoprecipitation assay revealed that more IRF8 bound to the IFN-stimulated response element of the Ifit1 gene than to those of the Ifit2 and the Ifit3 genes. Mutational analyses of IRF8 showed that its ability to bind DNA, interact with other proteins, and undergo sumoylation were all necessary to selectively repress Ifit1 gene induction in response to IFN. Our study revealed a new role for IRFs in differentially regulating the induction patterns of closely related IFN-stimulated genes that are located adjacent to one another in the mouse genome.

A Naturally Occurring Repeat Protein with High Internal Sequence Identity Defines a New Class of TPR-like Proteins

Linear repeat proteins often have high structural similarity and low (∼25%) pairwise sequence identities (PSI) among modules. We identified a unique P. anserina (Pa) sequence with tetratricopeptide repeat (TPR) homology, which contains longer (42 residue) repeats (42PRs) with an average PSI >91%. We determined the crystal structure of five tandem Pa 42PRs to 1.6 Å, and examined the stability and solution properties of constructs containing three to six Pa 42PRs. Compared with 34-residue TPRs (34PRs), Pa 42PRs have a one-turn extension of each helix, and bury more surface area. Unfolding transitions shift to higher denaturant concentration and become sharper as repeats are added. Fitted Ising models show Pa 42PRs to be more cooperative than consensus 34PRs, with increased magnitudes of intrinsic and interfacial free energies. These results demonstrate the tolerance of the TPR motif to length variation, and provide a basis to understand the effects of helix length on intrinsic/interfacial stability.

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader.

The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

An Unexpected Duo: Rubredoxin Binds Nine TPR Motifs to Form LapB, an Essential Regulator of Lipopolysaccharide Synthesis

Lipopolysaccharide (LPS) synthesis and export are essential pathways for bacterial growth, proliferation, and virulence. The essential protein LapB from Escherichia coli has recently been identified as a regulator of LPS synthesis. We have determined the crystal structure of LapB (without the N-terminal transmembrane helix) at 2Å resolution using zinc single-wavelength anomalous diffraction phasing derived from a single bound zinc atom. This structure demonstrates the presence of nine tetratricopeptide repeats (TPR) motifs, including two TPR folds that were not predicted from sequence, and a rubredoxin-type metal binding domain. The rubredoxin domain is bound intimately to the TPR motifs, which has not been previously observed or predicted. Mutations in the rubredoxin/TPR interface inhibit invivo cell growth, and invitro studies indicate that these modifications cause local displacement of rubredoxin from its binding site without changing the secondary structure of LapB. LapB is the first reported structure to contain both a rubredoxin domain and TPR motifs.

The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU(TPR)) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLU(TPR) at 2.4-Å resolution. Three non-canonical TPR motifs of each FLU(TPR) form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU(TPR)-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN.

The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q)XEX4-5(E/D/Q)X1-2(K/R)X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4.

Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.

Structural requirement of Ntc77 for spliceosome activation and first catalytic step.

The Prp19-associated complex is required for spliceosome activation by stabilizing the binding of U5 and U6 on the spliceosome after the release of U4. The complex comprises at least eight proteins, among which Ntc90 and Ntc77 contain multiple tetratricopeptide repeat (TPR) elements. We have previously shown that Ntc90 is not involved in spliceosome activation, but is required for the recruitment of essential first-step factor Yju2 to the spliceosome. We demonstrate here that Ntc77 has dual functions in both spliceosome activation and the first catalytic step in recruiting Yju2. We have identified an amino-terminal region of Ntc77, which encompasses the N-terminal domain and the first three TPR motifs, dispensable for spliceosome activation but required for stable interaction of Yju2 with the spliceosome. Deletion of this region had no severe effect on the integrity of the NTC, binding of NTC to the spliceosome or spliceosome activation, but impaired splicing and exhibited a dominant-negative growth phenotype. Our data reveal functional roles of Ntc77 in both spliceosome activation and the first catalytic step, and distinct structural domains of Ntc77 required for these two steps.

Crystal structure of N-terminal IFIT3 reveals domain-swapping dimerization

IFIT proteins are interferon-inducible, antiviral effectors that form a multiprotein complex with the ability to recognize markers of viral infection and subsequently restrict viruses. IFIT1, IFIT2 and IFIT3 are at the heart of the complex, interacting with each other and several host factors, forming what is known as the 'IFIT interactome'. Central to their ability to mediate complex formation is the tetratricopeptide repeat (TPR) motif, a general protein-protein interaction module comprising a pair of antiparallel alpha helices. Additionally The TPR motifs of IFIT proteins have the unique ability to recognize RNA. Whereas IFIT1 interacts with virus derived ssRNA, IFIT2 has been shown to interact with dsRNA; IFIT3 is not known to bind RNA. Importantly, structural information is available for the N-terminal domain of IFIT1 and full-length IFIT2, but not for IFIT3. To gain insight into the mechanisms regulating complex formation, we are targeting the structure of human IFIT3 before incorporation into the IFIT complex. To that end, we have determined a low resolution crystal structure of N-terminal IFIT3, which reveals a domain swapped dimer. Notably, IFIT3 dimerization is similar to IFIT2, but distinct from IFIT1, which dimerizes via its C-terminus. Sequence conservation and structural analysis suggest that IFIT2 and IFIT3 evolved a similar mechanism for domain swapping. We propose that IFIT2 and IFIT3 may interact by forming domain swapped heterodimers. Current work is aimed at investigating the mechanisms of domain swapping via mutational analysis, and determining the structure of C-terminal human IFIT3.

BB0238, a presumed tetratricopeptide repeat-containing protein, is required during Borrelia burgdorferi mammalian infection.

The Lyme disease spirochete, Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle of B. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens. bb0238 encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role of bb0238 during mammalian infection, a bb0238-deficient mutant was constructed. The bb0238 mutant was attenuated in mice infected via needle inoculation, and complementation of bb0238 expression restored infectivity to wild-type levels. bb0238 expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed under in vitro conditions. bb0238 is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection. B. burgdorferi clones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in the bb0238 deletion mutant, suggesting that BB0238 may contain a functional TPR domain.