Abstract
Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs.
Highlights
Ancient globins such as neuroglobin (Ngb), globin X and androglobin are endowed with heme iron hexacoordination, whereas pentacoordinated myoglobins (Mb) and hemoglobins (Hb), characterized by a complex respiratory role, appeared later in evolution
Traces were fitted as single (WT and Gly-loop, Fig. 1A) or double exponentials (F106A and Gly-loop/F106A, Fig. 1A)
As observed previously[20], CO binding to Wild type (WT) Ngb exhibits a mono-phasic decay at 426 nm, and so does the Gly-loop mutant
Summary
Ancient globins such as neuroglobin (Ngb), globin X and androglobin are endowed with heme iron hexacoordination, whereas pentacoordinated myoglobins (Mb) and hemoglobins (Hb), characterized by a complex respiratory role, appeared later in evolution. The dissociation of His[64] is the rate-limiting step for ligand binding and it triggers a repositioning of the heme which slides further inside a large internal cavity This movement is associated with the motion of helices and loops[14,15,16], and probably confers to Ngb a more stable conformation. Molecular dynamics (MD) simulations supported a His[64] gating out movement coupled with a motion of the CD corner upon ligand binding, larger than the one described by crystallography[17,18] Another structural feature of neuroglobin is the double insertion of the heme with 70:30 proportion[14]. We probed the role of heme sliding in the internal cavity, facilitated by reducing the bulk in position 106 and the role of the CD loop in governing ligand affinity and hexacoordination
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