Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures.

Simon Boje Hansen,Nick Stub Laursen,Gregers Rom Andersen,Kasper R Andersen

doi:10.1107/s2059798317013171

Simon Boje Hansen, Nick Stub Laursen + Show 2 more

Open Access

https://doi.org/10.1107/s2059798317013171

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Abstract

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.

Highlights

The production of well diffracting protein crystals is a major challenge in macromolecular X-ray crystallography
We describe the introduction of cysteine residues at conserved framework serine positions in a Nb specific for human complement component C5 and subsequent site-specific labelling with Hg derivatives and structure determination by single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS)
It was our expectation that the crystallization of a monomeric para-chloromercuribenzoic acid (PCMB)-derivatized Nb would reveal structures with a Hg atom inserted between the benzoate moiety and the side chain of Cys85

Summary

Introduction

The production of well diffracting protein crystals is a major challenge in macromolecular X-ray crystallography. Large multi-domain proteins and membrane proteins are inherently difficult to crystallize owing to conformational heterogeneity and the lack of suitable surface chemistry that allows the formation of a crystal lattice. Crystallization chaperones are auxiliary proteins that increase the chance of crystallization by reducing conformational flexibility and providing well ordered surfaces to form crystal lattice contacts. Monoclonal antibody Fab fragments derived from IgG are the most widely used chaperones (Uysal et al, 2009) and may simultaneously provide phase information for structure determination by molecular replacement. Several alternative chaperones have been developed, including DARPins, single-chain variable fragments and nanobodies (Nbs; Pardon et al, 2014). Nbs are derived from natural llama heavy-chain antibodies that are devoid of a light chain and in which the heavy-chain variable domain (VHH) exclusively mediates the interaction with the antigen (Muyldermans, 2013).

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