A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops

AbstractBackground: Camelid serum contains a large fraction of functional heavy-chain antibodies – homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor.Results: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05–RNase A complex has been solved at 2.8 Å. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies.Conclusions: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction

Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques

Protein crystallisation under microgravity conditions: What did we learn on TIM crystallisation from the Soyuz missions?

The protein Triose Phosphate Isomerase from the hyperthermophilic organism Thermotoga maritima was crystallised on board of the International Space Station in the framework of the Soyuz missions. In this paper we report on the scientific results obtained during these flights. Firstly it was shown that different crystallisation techniques and environments can result in different crystal forms for the same protein in the same crystallisation conditions, what is presumably due to a change in the rate at which supersaturation is achieved. Secondly, the X-ray quality of the crystals grown in the ISS is superior to their ground control crystals. Mimicking microgravity on ground, by adding a small amount of gel to avoid convection, also results in an improvement of X-ray quality. Nevertheless our analysis shows that the crystals obtained in this gelled ground environment are of inferior quality as compared to their space homologues. Finally we observed movement of crystals grown in the International Space Station, not only because of g-jitters but also due to residual accelerations. This has an important effect on concentration gradients of precipitants and therefore on the solubility of the protein

A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues(1-3), often with serious pathological consequences. Non-neuropathic systemic amyloidosis (4-6) is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody(7-9) raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases