(a) Chemical structure of allosteric inhibitors. (b) Visualization of the VCP structure and the amino acids modified by the azido derivatives. Top, schematic representation of the three-dimensional hexameric structure derived from PDB code 3CF1 (left), showing the allosteric binding site (right). Blue, green and pink are used to distinguish the N, D1 and D2 domains of individual protomers in the hexameric structure, respectively. Different shades distinguish adjacent protomers. The region of binding identified by photoaffinity labeling experiments is highlighted. The right panel shows in detail the lateral tunnel formed by two D1 domains and one D2 domain of two adjacent protomers leading to the central pore of the hexamer. The position of the Lys615 (red) and Asn616 (yellow) residues in D2, derivatized by the azido probes, is highlighted. Bottom, modeled binding site of allosteric inhibitors and one example of a superimposed azide derivative (NMS-454) in close proximity to the reacting amino acid side chain (Asn616). The azido moiety was added after optimizing the orientation of the compound in the unmodified three-dimensional structure. The binding site is situated in a region connecting one nucleotide pocket (N protomer) to the next (N + 1 protomer) by means of a close interaction between the ISS motif residue Asp609 and the second region of homology (SRH) residue Arg638. Additional amino acids involved in the mutation studies are also shown. 