Atomic resolution structure of biotin-free Tyr43Phe streptavidin: what is in the binding site?

The streptavidin–biotin system is an example of a high-affinity protein–ligand pair (K_a ≃ 10¹³ mol⁻¹). The thermodynamic and structural properties have been extensively studied as a model system for protein–ligand interactions. Here, the X-ray crystal structure of a streptavidin mutant of a residue hydrogen bonding to biotin [Tyr43Phe (Y43F)] is reported at atomic resolution (1.14 Å). The biotin-free structure was refined with anisotropic displacement parameters (using the SHELXL97 program package). The high-resolution data also allowed interpretation of side-chain and residue disorder in 41 residues where alternate conformations were refined. The Y43F mutation is unambiguously observed in difference maps, although only a single O atom per monomer is altered. The atomic resolution enabled the identification of 2-methyl-2,4-pentanediol (MPD) molecules in the biotin-binding pocket for the first time. Electron density for MPD was observed in all four subunit binding sites of the tetrameric protein. This was not possible with data at lower resolution (1.8–2.3 Å) for wild-type streptavidin or mutants in the same crystal form using MPD in the crystallization. The impact of MPD binding on these studies is discussed.