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The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys-Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody-protein complexes. [ABSTRACT FROM AUTHOR]

The integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMβ2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside-inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMβ2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMβ2-specific agonist. Taken together, our data suggest that the iC3b-αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMβ2. [ABSTRACT FROM AUTHOR]

P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (P IB -ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, P IB -ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting P IB -ATPase ZntA from Shigella sonnei (SsZntA), aiming at developing tools to assist the characterization of the structure and function of this class of transporters. We identify six different Nbs that bind detergent stabilized SsZntA. We further assess the effect of the Nbs on the catalytic function of SsZntA, and find that five nanobodies associate without affecting the function, while one nanobody significantly reduces the ATPase activity. This study paves the way for more refined mechanistical and structural studies of zinc-transporting P IB -ATPases.