Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.

Kamel El Omari,Maike Bublitz,Vitaliy Mykhaylyk,Armin Wagner,Konstantinos Beis,Ramona Duman,Claus Olesen,Nada Mohamad,Kiran Bountra,Hok-Sau Kwong,Katja Schlegel,Maria Romano,Jesper Vuust Moller

doi:10.1107/s2052252520012312

Abstract

The structure determination of soluble and membrane proteins can be hindered by the crystallographic phase problem, especially in the absence of a suitable homologous structure. Experimental phasing is the method of choice for novel structures; however, it often requires heavy-atom derivatization, which can bedifficult and time-consuming. Here, a novel and rapid method to obtain experimental phases for protein structure determination by vanadium phasing is reported. Vanadate is a transition-state mimic of phosphoryl-transfer reactions and it has the advantage of binding specifically to the active site of numerous enzymes catalyzing this reaction. The applicability of vanadium phasing has been validated by determining the structures of three different protein-vanadium complexes, two of which are integral membrane proteins: the rabbit sarcoplasmic reticulum Ca2+-ATPase, the antibacterial peptide ATP-binding cassette transporter McjD from Escherichia coli and the soluble enzyme RNAse A from Bos taurus. Vanadium phasing was successful even at low resolution and despite severe anisotropy in the data. This method is principally applicable to a large number of proteins, representing six of the seven Enzyme Commission classes. It relies exclusively on the specific chemistry of the protein and it does not require any modifications, making it a very powerful addition to the phasing toolkit. In addition to the phasing power of this technique, the protein-vanadium complexes also provide detailed insights into the reaction mechanisms of the studied proteins.

Highlights

Membrane proteins mediate essential processes in all living organisms, including signaling, nutrient uptake, xenobiotic efflux and multidrug resistance
This study demonstrates the successful use of vanadium Single-wavelength anomalous diffraction (SAD) phasing to determine three protein–vanadate complex structures, two of which are membrane-protein representatives from the ATP-binding cassette (ABC) transporter and P-type ATPase families, the structural determination of which had previously been dependent on molecular replacement
Finding the vanadium sites does not necessarily warrant successful structure determination; phenix.anomalous_signal predicted a low FOM* for McjD and SERCA (0.3 and 0.2, respectively) and an FOM* of 0.4 for RNAse A, the latter likely to be owing to the higher resolution of the ribonuclease A (RNAseA) data set

Summary

Introduction

Membrane proteins mediate essential processes in all living organisms, including signaling, nutrient uptake, xenobiotic efflux and multidrug resistance. Membrane proteins account for 20–30% of the genome and many of them are important drug targets. The fact that membrane proteins reside within the lipid bilayer makes them notoriously difficult proteins to work with. Besides the difficulties encountered during protein expression, purification and crystallization (Carpenter et al, 2008), membrane-protein crystals tend to diffract to low resolution and often suffer from high mosaicity and nonisomorphism, rendering structure determination difficult if molecular replacement is not an option. 7, 1092–1101 research papers amongst the 50 000 distinct proteins in the Protein Data Bank (wwPDBconsortium, 2019), less than one thousand are membrane proteins (https://blanco.biomol.uci.edu/mpstruc/). Considering that more than 60% of drug targets are membrane proteins (Overington et al, 2006), there is a strong unmet need to facilitate their structure determination

Methods

Results

Conclusion