Phosphonated Calixarene as a “Molecular Glue” for Protein Crystallization

Protein crystallization remains a serious bottleneck to structure determination by X-ray diffraction methods. Compounds acting as “molecular glue” provide a promising strategy to overcome this bottleneck. Such molecules interact via noncovalent bonds with two or more protein surfaces to promote lattice formation. Here, we report a 1.5 Å resolution crystal structure of lysine-rich cytochrome <i>c</i> complexed with <i>p</i>-phosphonatomethyl-calix­[4]­arene (<b>pmclx</b>_<b>4</b>). Evidence for complex formation in solution was provided by NMR studies. Similar to <i>p</i>-sulfonato-calix­[4]­arene (<b>sclx</b>_<b>4</b>), the cavity of <b>pmclx</b>_<b>4</b> entrapped a single lysine side chain. Interesting features of protein recognition by the phosphonate substituents were identified in the crystal structure. A new calixarene binding site was identified at Lys54. The electron density at this site indicated two distinct calixarene conformers, suggesting a degree of ligand mobility. The role of <b>pmclx</b>_<b>4</b> in protein crystal packing (molecular glue and patchy particle model) as well as differences in protein-binding with respect to <b>sclx</b>_<b>4</b> are discussed