Straight-chain alkyl isocyanides open the distal histidine gate in crystal structures of myoglobin

Abstract

Crystal structures of methyl, ethyl, propyl, and butyl isocyanide bound to sperm whale myoglobin (Mb) reveal two major conformations. In the in conformer, His(E7) is in a "closed" position, forcing the ligand alkyl chain to point inward. In the out conformer, His(E7) is in an "open" position, allowing the ligand side chain to point outward. A progressive increase in the population of the out conformer is observed with increasing ligand length in P2(1) crystals of native Mb at pH 7.0. This switch from in to out with increasing ligand size also occurs in solution as measured by the decrease in the relative intensity of the low-frequency ( approximately 2075 cm(-1)) versus high-frequency ( approximately 2125 cm(-1)) isocyano bands. In contrast, all four isocyanides in P6 crystals of wild-type recombinant Mb occupy the in conformation. However, mutating either His64 to Ala, creating a "hole" to solvent, or Phe46 to Val, freeing rotation of His64, causes bound butyl isocyanide to point completely outward in P6 crystals. Thus, the unfavorable hindrance caused with crowding a large alkyl side chain into the distal pocket appears to be roughly equal to that for pushing open the His(E7) gate and is easily affected by crystal packing. This structural conclusion supports the "side path" kinetic mechanism for O(2) release, in which the dissociated ligand first moves toward the protein interior and then encounters steric resistance, which is roughly equal to that for escaping to solvent through the His(E7) channel.

Figure 1

X-ray crystal structures of native Mb CNC1 through CNC4 (top row) and wt Mb CNC1 through -CNC4 (bottom row). For native Mb, in P2₁ crystals at pH 7.0, an increase in the isocyanide length corresponds to a greater occupancy of the open conformation of His64 (orange 2F_o-F_c electron density surface) and the out conformation of the ligand (blue 2F_o-F_c electron density surface). However, for wt Mb in P6 crystals at pH 9.0, little conformational variation with CNR chain length is observed. Stick representations show the heme (white) and key amino acid side chains (CPK with yellow carbons) of the refined structures. 2F_o-F_c electron density surfaces are shown in orange, blue, and gray for the His64, ligand, and other side chains, respectively.

Figure 2

A. Dependence of the His64 conformation on the CNR length and pH for native Mb in the P2₁ crystal structure. The 2F_o-F_c and stick color schemes are given in Fig. 1. B. The conformations of MbCNRs in solution are relatively invariant with pH between ~6 and 9.

Figure 3

A. The effects of mutations that reduce steric hindrance by His64 on bound CNC4. In P6 crystals at pH 9.0, the bound ligand changed from the in conformation for wt MbCNC4 to the out conformation for both the H64A (bottom left) and F46V (bottom right) MbCNC4 complexes. B. The effects of the same mutations on the ν_CN bands in FTIR spectra. The IR spectra were collected at pH 7.0 in 0.1 M phosphate buffer.

Figure 4

The effect of the crystal form (P2₁ and P6) on the distal pocket structures of MbCO and MbCNC4. A. The binding pocket residues and heme groups of Mb structures aligned by all protein Cα atoms, including native Mb (P2₁ crystals, pH ~7.0) complexed to CO (light blue; PDB IDs 1bzr, 1vxf and 1mbc) and CNC4 (dark blue; this work), and wt Mb (P6 crystals, pH 9.0) complexed to CO (red; PDB IDs 2mgk and 1jw8) and CNC4 (black; this work). The ligands are not shown. B. The same structures were aligned by superimposing the atoms of the heme plane. This alignment emphasizes crystal lattice-dependent variations in the positions of the distal pocket amino acid side chains relative to the ligand heme complex. C. Intermonomer contacts and sulfate binding at the CD-corner of Mb that are present in P2₁ but not P6 crystals. The structures, colors and alignment are as in panel A. Spheres are shown with van der Waals radii for the native MbCNC4 Thr51-Cγ; Asp60-Cγ, Ala53-Cβ, His64-Cε, Arg45-O', -Nη1 and -Nη2, and sulfate atoms, and for the wt MbCNC4 Asp60-Cγ and heme propionate O1D atoms.