Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

Abstract

-wavelength anomalous dispersion; SAD; single isomorphous replacement with anomalous signal; SIRAS.PDB references: Nb36-Nat1, 5nlu; Nb36-Nat2, 5nlw; Nb36-C85-1, 5nm0; Nb36-C85-2, 5nml 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 radiationinduced 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.

A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

“…Compared with existing C3 inhibitors, hC3Nb1 is easy to produce as a recombinant protein; it can be tagged for immobilization, the W102A variant is available as a negative control, and like other nanobodies it may be fused with itself or other proteins to confer multivalent binding and a highly specific in vivo distribution. We and others have also shown that Nbs may be engineered to contain free cysteines that are reactive with fluorescent dyes, thereby allowing their use in applications like flow cytometry, in vivo imaging, and fluorescence microscopy (36,37). The site-specific introduction of a biotin molecule used here for SPR immobilization also opens for a wealth of applications based on the biotin-streptavidin interaction.…”

Structural signatures in EPR3 define a unique class of plant carbohydrate receptors

“…We therefore generated a miniature llama-derived antibody (nanobody) to facilitate crystallisation. We raised an immune response against EPR3 by immunising a llama and selected nanobodies by phage display 12,13 . The high-affinity nanobody, Nb186, forms a stable complex with EPR3 as demonstrated by a mobility shift in size-exclusion chromatography (SEC) experiments (Supplementary Fig.…”

Antigen footprint governs activation of the B cell receptor

“…For DNA-PAINT super-resolution microscopy, the serine residue at position 87 (Ser87) was replaced with a cysteine residue (Cys87) for site-specific labelling of a DNA oligonucleotide. This design was based on the study by Hansen et al 60 , who obtained high conjugation efficiency with engineered cysteine residues slightly distant from the C-terminus.…”