Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Three-dimensional structure of the diphtheria toxin repressor in complex with divalent cation co-repressors

AbstractBackground: When Corynebacterium diphtheriae encounters an environment with a low concentration of iron ions, it initiates the synthesis of several virulence factors, including diphtheria toxin. The diphtheria toxin repressor (DtxR) plays a key role in this iron–dependent, global regulatory system and is the prototype for a new family of iron –dependent repressor proteins in Gram-positive bacteria. This study aimed to increase understanding of the general regulatory principles of cation binding to DtxR.Results The crystal structure of dimeric DtxR holo-repressor in complex with different transition metals shows that each subunit comprises an amino-terminal DNA–binding domain, an interface domain (which contains two metal-binding sites) and a third, very flexible carboxy-terminal domain. Each DNA–binding domain contains a helix–turn–helix motif and has a topology which is very similar to catabolite gene activator protein (CAP). Molecular modeling suggests that bound DNA adopts a bent conformation with helices α3 of DtxR interacting with the major grooves. The two metal-binding sites lie ∼10 Å apart. Binding site 2 is positioned at a potential hinge region between the DNA–binding and interface domains. Residues 98–108 appear to be crucial for the functioning of the repressor; these provide four of the ligands of the two metal-binding sites and three residues at the other side of the helix which are at the heart of the dimer interface.Conclusion The crystal structure of the DtxR holo-repressor suggests that the divalent cation co–repressor controls motions of the DNA-binding domain. In this way the metal co–repressor governs the distance between operator recognition elements in the two subunits and, consequently, DNA recognition