A disulfide-free single-domain V(L) intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition.

We present the crystal structure and biophysical characterization of a human V(L) [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has been engineered for antigen recognition in the absence of its intradomain disulfide bond, otherwise conserved in the Ig fold. Analytical ultracentrifugation demonstrated that the αHtt-V(L) 12.3 domain is a stable monomer under physiological conditions even at concentrations >20 µM. Using peptide SPOT arrays, we identified the minimal binding epitope to be EKLMKAFESLKSFQ, comprising the N-terminal residues 5-18 of Htt and including the first residue of the poly-Gln stretch. X-ray structural analysis of αHtt-V(L) both as apo protein and in the presence of the epitope peptide revealed several interesting insights: first, the role of mutations acquired during the combinatorial selection process of the αHtt-V(L) 12.3 domain-initially starting from a single-chain Fv fragment-that are responsible for its stability as an individually soluble Ig domain, also lacking the disulfide bridge, and second, a previously unknown mode of antigen recognition, revealing a novel paratope. The Htt epitope peptide adopts a purely α-helical structure in the complex with αHtt-V(L) and is bound at the base of the complementarity-determining regions (CDRs) at the concave ß-sheet that normally gives rise to the interface between the V(L) domain and its paired V(H) (variable domain Ig heavy chain) domain, while only few interactions with CDR-L1 and CDR-L3 are formed. Notably, this noncanonical mode of antigen binding may occur more widely in the area of in vitro selected antibody fragments, including other Ig-like scaffolds, possibly even if a V(H) domain is present.

Structural and biochemical analyses reveal a monomeric state of the bacterial lipocalin Blc.

The first bacterial member of the lipocalin protein family, Blc, was identified in Escherichia coli as an outer membrane lipoprotein that is expressed under conditions of environmental stress. Previous crystallographic studies in space group P212121 with two molecules per asymmetric unit, supported by static light-scattering experiments in solution, indicated that Blc may form a functional homodimer with lysophospholipid binding activity. Here, a new crystal structure of recombinant Blc in space group I4122 with one molecule in the asymmetric unit is described. The crystal packing differs considerably from that observed previously, which was determined using an N-terminally extended version of Blc dubbed `Blc-X'. In particular, the characteristic large interface region that was previously described as being responsible for stable dimer formation is absent in the I4122 crystal structure. Thus, the dimerization behaviour of Blc-X was most likely to be caused by the additional N-terminal peptide segment resulting from the cloning strategy employed. In contrast, we used a native-like N-terminus for Blc with just the lipid-anchored first Cys residue replaced by Ala. The fully monomeric status of this recombinant version of Blc in solution was confirmed by size-exclusion chromatography as well as analytical ultracentrifugation. Consequently, these data shed new light on the previously postulated lipid-binding mechanism and biological role of Blc. Beyond this, our findings illustrate that cloning artefacts, which frequently result from recombinant protein production for structural studies, must be considered with special caution when interpreting oligomerization and/or conformational effects.

A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavity.

Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P2(1) with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6 A resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P2(1) crystal form uncovered major conformational changes (i) in beta-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the beta-barrel and (iii) in the extended C-terminal segment, which is attached to the beta-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family.

Structure of lumazine protein, an optical transponder of luminescent bacteria.

The intensely fluorescent lumazine protein is believed to be involved in the bioluminescence of certain marine bacteria. The sequence of the catalytically inactive protein resembles that of the enzyme riboflavin synthase. Its non-covalently bound fluorophore, 6,7-dimethyl-8-ribityllumazine, is the substrate of this enzyme and also the committed precursor of vitamin B(2). An extensive crystallization screen was performed using numerous single-site mutants of the lumazine protein from Photobacterium leiognathi in complex with its fluorophore and with riboflavin, respectively. Only the L49N mutant in complex with riboflavin yielded suitable crystals, allowing X-ray structure determination to a resolution of 2.5 A. The monomeric protein folds into two closely similar domains that are structurally related by pseudo-C(2) symmetry, whereby the entire domain topology resembles that of riboflavin synthase. Riboflavin is bound to a shallow cavity in the N-terminal domain of lumazine protein, whereas the C-terminal domain lacks a ligand.

The carbohydrate recognition domain of Langerin reveals high structural similarity with the one of DC-SIGN but an additional, calcium-independent sugar-binding site.

Langerin is a type II transmembrane oligosaccharide receptor on Langerhans cells (LCs), a prominent subclass of dendritic cells (DCs) that mediate immune responses in epithelia and play a role in HIV degradation. Its extracellular moiety comprises a neck region with several heptad repeats and an exposed carboxy-terminal calcium-type carbohydrate-recognition domain (CRD). The CRD of human Langerin, which was expressed as a soluble protein in the periplasm of E. coli, was crystallized both alone and in the presence of two sugars, followed by X-ray analyses to resolutions of 2.5A for apo-Langerin and to 1.6A and 2.1A for the complexes with mannose and maltose, respectively. The fold of the Langerin CRD (dubbed LangA) resembles that of other typical C-type lectins such as DC-SIGN. However, especially in the long loop region (LLR), which is responsible for carbohydrate-binding, two additional secondary structure elements are present: a 3(10) helix and a small beta-sheet arising from the extended beta-strand 2, which enters into a hairpin and a new strand beta2'. Unexpectedly, the crystal structures in the presence of maltose and mannose reveal two sugar-binding sites. One is calcium-dependent and structurally conserved in the C-type lectin family whereas the second one represents a novel, calcium-independent type. Based on these data, a model for the binding of mannan, a component of many endogenous as well as viral glycoproteins, is proposed and the differences in binding behavior between Langerin and DC-SIGN with respect to the Lewis X carbohydrate antigen and its derivatives can be explained. Therefore, the crystal structure of LangA should be helpful for the development of new marker reagents selective for LCs and also of therapeutic compounds that may enhance the inhibitory role of Langerin towards HIV infection.

Biochemical characterization of the recombinant human Nogo-A ectodomain.

Nogo-A is a physiologically relevant inhibitor of neuronal growth and regeneration in the myelin of the adult human central nervous system and has attracted considerable attention as a molecular target for the treatment of spinal cord injuries. To gain insight into the structural and functional properties of the large extramembrane region that is characteristic for the Nogo-A splice form of this member of the Reticulon family of membrane proteins, we cloned and expressed the region comprising residues 334-966 as a soluble homogeneous protein in the periplasm of Escherichia coli. SDS/PAGE, under nonreducing conditions, and a systematic substitution analysis of all six Cys residues of Nogo-A indicated that this domain forms two structural disulfide bonds among Cys residues 424, 464, 559 and 597, whereas the Cys residues at positions 699 and 912 seem to be dispensable for folding. The occurrence of a hot spot for host cell proteases and a limited proteolysis experiment suggest that the N-terminal region of Nogo-A up to residue 373 is structurally disordered. Although analytical gel permeation chromatography revealed a large apparent molecular size for the recombinant Nogo-A fragment, indicating oligomer formation, data from analytical ultracentrifugation and dynamic light scattering support a stable monomeric quaternary structure. Notably, the CD spectrum is indicative of a high content of random coil, such that Nogo-A exhibits--at least in part--features of a natively unfolded protein. Nevertheless, the protein fragment identified in this study, as well as its biochemical analysis, provide a promising starting point for future investigations to track down globular subdomains and functionally important regions as well as putative receptor-binding sites therein.

Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii.

The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.