Molecular recognition requires dimerization of a VHH antibody.

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

Hairpin RNA-induced conformational change of a eukaryotic-specific lysyl-tRNA synthetase extension and role of adjacent anticodon-binding domain.

Human lysyl-tRNA synthetase (hLysRS) is essential for aminoacylation of tRNALys Higher eukaryotic LysRSs possess an N-terminal extension (Nterm) previously shown to facilitate high-affinity tRNA binding and aminoacylation. This eukaryote-specific appended domain also plays a critical role in hLysRS nuclear localization, thus facilitating noncanonical functions of hLysRS. The structure is intrinsically disordered and therefore remains poorly characterized. Findings of previous studies are consistent with the Nterm domain undergoing a conformational transition to an ordered structure upon nucleic acid binding. In this study, we used NMR to investigate how the type of RNA, as well as the presence of the adjacent anticodon-binding domain (ACB), influences the Nterm conformation. To explore the latter, we used sortase A ligation to produce a segmentally labeled tandem-domain protein, Nterm-ACB. In the absence of RNA, Nterm remained disordered regardless of ACB attachment. Both alone and when attached to ACB, Nterm structure remained unaffected by titration with single-stranded RNAs. The central region of the Nterm domain adopted α-helical structure upon titration of Nterm and Nterm-ACB with RNA hairpins containing double-stranded regions. Nterm binding to the RNA hairpins resulted in CD spectral shifts consistent with an induced helical structure. NMR and fluorescence anisotropy revealed that Nterm binding to hairpin RNAs is weak but that the binding affinity increases significantly upon covalent attachment to ACB. We conclude that the ACB domain facilitates induced-fit conformational changes and confers high-affinity RNA hairpin binding, which may be advantageous for functional interactions of LysRS with a variety of different binding partners.

Structural basis for collagen recognition by the immune receptor OSCAR.

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 µM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

Recombinant soluble CD137 prevents type one diabetes in nonobese diabetic mice.

Nonobese diabetic (NOD) mice are genetically programmed to spontaneously develop type one diabetes (T1D). Multiple Insulin dependent diabetes (Idd) genetic loci have been identified but their functional effects are mostly poorly understood. TnfsfR9, expressing the protein product CD137, is a strong candidate gene in the Idd9.3 locus, and NOD.B10 Idd9.3 mice are significantly protected from type one diabetes (T1D). We previously showed that nonobese diabetic (NOD) mice have a deficiency in the numbers of CD137(pos) T regulatory cells, that CD137(pos) Tregs are the source of soluble CD137 (sCD137), and that NOD mice have low serum levels of sCD137. To test the hypothesis that correcting low levels of sCD137 could affect the disease, we constructed a lentiviral vector producing recombinant sCD137; this physiologic sCD137 is glycosylated and exists primarily as a dimer. NOD mice treated with the recombinant sCD137 are protected from developing T1D. Insulitis is significantly decreased, but not eliminated in the sCD137 treated mice, however insulin producing pancreatic beta cells are preserved despite residual insulitis. To begin to understand the protective immune mechanisms of sCD137, we tested sCD137 in vitro. It was previously suggested that sCD137 simply blocked the interaction between CD137 (on T cells) and CD137 ligand (on antigen presenting cells (APCs)). Here however, we use an APC independent assay and demonstrate that sCD137 can actively suppress highly purified CD4 T cells in a CD137L dependent fashion. These results support the hypothesis that sCD137 acts in a negative feedback loop to actively suppress over-zealous immune responses, and that it can be used clinically to suppress autoimmunity. sCD137 is an important Treg derived natural immunosuppressive molecule that regulates effector T cells to avert diabetes in vivo.

Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA.

Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).

Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino terminus.

The AsnC/Lrp family of regulatory proteins links bacterial and archaeal transcription patterns to metabolism. In Escherichia coli, Lrp regulates approximately 400 genes, over 200 of them directly. In earlier studies, lrp genes from Vibrio cholerae, Proteus mirabilis, and E. coli were introduced into the same E. coli background and yielded overlapping but significantly different regulons. These differences were seen despite amino acid sequence identities of 92% (Vibrio) and 98% (Proteus) to E. coli Lrp, including complete conservation of the helix-turn-helix motifs. The N-terminal region contains many of the sequence differences among these Lrp orthologs, which led us to investigate its role in Lrp function. Through the generation of hybrid proteins, we found that the N-terminal diversity is responsible for some of the differences between orthologs in terms of DNA binding (as revealed by mobility shift assays) and multimerization (as revealed by gel filtration, dynamic light scattering, and analytical ultracentrifugation). These observations indicate that the N-terminal tail plays a significant role in modulating Lrp function, similar to what is seen for a number of other regulatory proteins.

Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives.

The bacteriophage T4 encodes 10 proteins, known collectively as the replisome, that are responsible for the replication of the phage genome. The replisomal proteins can be subdivided into three activities; the replicase, responsible for duplicating DNA, the primosomal proteins, responsible for unwinding and Okazaki fragment initiation, and the Okazaki repair proteins. The replicase includes the gp43 DNA polymerase, the gp45 processivity clamp, the gp44/62 clamp loader complex, and the gp32 single-stranded DNA binding protein. The primosomal proteins include the gp41 hexameric helicase, the gp61 primase, and the gp59 helicase loading protein. The RNaseH, a 5' to 3' exonuclease and T4 DNA ligase comprise the activities necessary for Okazaki repair. The T4 provides a model system for DNA replication. As a consequence, significant effort has been put forth to solve the crystallographic structures of these replisomal proteins. In this review, we discuss the structures that are available and provide comparison to related proteins when the T4 structures are unavailable. Three of the ten full-length T4 replisomal proteins have been determined; the gp59 helicase loading protein, the RNase H, and the gp45 processivity clamp. The core of T4 gp32 and two proteins from the T4 related phage RB69, the gp43 polymerase and the gp45 clamp are also solved. The T4 gp44/62 clamp loader has not been crystallized but a comparison to the E. coli gamma complex is provided. The structures of T4 gp41 helicase, gp61 primase, and T4 DNA ligase are unknown, structures from bacteriophage T7 proteins are discussed instead. To better understand the functionality of T4 DNA replication, in depth structural analysis will require complexes between proteins and DNA substrates. A DNA primer template bound by gp43 polymerase, a fork DNA substrate bound by RNase H, gp43 polymerase bound to gp32 protein, and RNase H bound to gp32 have been crystallographically determined. The preparation and crystallization of complexes is a significant challenge. We discuss alternate approaches, such as small angle X-ray and neutron scattering to generate molecular envelopes for modeling macromolecular assemblies.