Ligand Uptake Modulation by Internal Water Molecules and Hydrophobic Cavities in Hemoglobins

Abstract

Internal water molecules play an active role in ligand uptake regulation, since displacement of retained water molecules from protein surfaces or cavities by incoming ligands can promote favorable or disfavorable effects over the global binding process. Detection of these water molecules by X-ray crystallography is difficult given their positional disorder and low occupancy. In this work, we employ a combination of molecular dynamics simulations and ligand rebinding over a broad time range to shed light into the role of water molecules in ligand migration and binding. Computational studies on the unliganded structure of the thermostable truncated hemoglobin from Thermobifida fusca (Tf-trHbO) show that a water molecule is in the vicinity of the iron heme, stabilized by WG8 with the assistance of YCD1, exerting a steric hindrance for binding of an exogenous ligand. Mutation of WG8 to F results in a significantly lower stabilization of this water molecule and in subtle dynamical structural changes that favor ligand binding, as observed experimentally. Water is absent from the fully hydrophobic distal cavity of the triple mutant YB10F-YCD1F-WG8F (3F), due to the lack of residues capable of stabilizing it nearby the heme. In agreement with these effects on the barriers for ligand rebinding, over 97% of the photodissociated ligands are rebound within a few nanoseconds in the 3F mutant case. Our results demonstrate the specific involvement of water molecules in shaping the energetic barriers for ligand migration and binding.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

“…The value of the rebinding rate k -1 is remarkably large compared to, for instance, rebinding to the highly reactive type II truncated Hb from Thermobifida fusca (TrHbO), for which rebinding occurs with a rate of 3 × 10 8 s -1 64 , a value which is 20 fold smaller than the one for NP7. Mutation of distal cavity residues capable of stabilizing water molecules nearby the binding site lowers the barrier for rebinding and increases the rate to 2 × 10 9 s -1 65 , a value which becomes comparable to the value for NP7 at neutral pH, but is still one order of magnitude smaller than observed for NP7 at pH 5.5.…”

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

“…Small ligand association in the trHb family is presumably regulated by two main processes: i) ligand migration from solvent bulk to the protein distal site cavity, ii) displacement of water molecules from the distal site cavity 15 – 17 , 35 . With this in mind, we performed classical MD simulations as they allow us to investigate both processes involved in ligand association.…”

“…In addition, the presence of a smaller residue at the G8 position in the Mt-trHbO mutant (WG8F) was observed to increase the small ligand association constant, although the molecular details of this process were not investigated 12 – 14 . It has also been noted that in myoglobin, M. Tuberculosis trHbN as well as in T. fusca trHbO, internal water molecules were observed to block the heme accessibility, thus delaying ligand binding 15 – 17 .…”

Lessons Learned from 50 Years of Hemoglobin Research: Unstirred and Cell-Free Layers, Electrostatics, Baseball Gloves, and Molten Globules

“…In Mb, the A-helix blocks any ligand movement out through the interior of the Mb structure, whereas it is missing in the smaller CerHb structure. Since the publication of our original Mb mapping study in 2001, a significant number of articles, including time-resolved crystallography experiments (92), work with larger isocyanide ligands (7,8,99), molecular dynamics simulations (9,14,15,54), and transition path theory (115), have been published supporting the E7 gate pathway in mammalian Mbs.…”