The histamine N-methyltransferase T105I polymorphism affects active site structure and dynamics

Abstract

Histamine N-methyltransferase (HNMT) is the primary enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels than the 105T protein. Crystal structures of both variants have been determined but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations for both 105T and 105I at 37 degrees C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened intramolecular interactions between residue 105 and residues within helix alpha3 and strand beta3. This altered, tighter packing is translated to the active site, resulting in the reorientation of several cosubstrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lowers the hydrophobicity of the substrate-binding site.

Figure 1. Bi-domain structure of histamine N-methyltransferase

(A) Ribbon diagram of HNMT (2AOT) with the SAM and histamine binding domains colored gray and red, respectively. Product S-adenosylhomocysteine (SAH), inhibitor diphenhydramine (2PM), and polymorphic residue 105 are colored by atom. (B) Key residues in the highly conserved SAM-dependent methyltransferase domain. SAM interacts with residues from α4 (Q94), α5 (S120), β2 (E89, P90), β3 (T119) and β4 (I142, M144). SAH is colored magenta and shown in licorice representation, residues are colored by atom and shown in space-filling representation. (C) Key residues in the histamine-binding domain. A trio of catalytic residues (E28, Q143 and N283) is involved in the N-methylation of HNMT substrates. The substrate is buried in a hydrophobic domain lined with aromatic residues (shown in licorice representation).

Figure 2. Mobility of HNMT during MD

(A) Cα-RMS fluctuations (Å) per residue from the 105T (green) and 105I (blue) MD simulations at 37°C. Cα-RMSFs were calculated relative to the average structure over the last 10 ns of each simulation. Experimental B-factors of the 105T HNMT crystal structure (2AOT, (28)) are colored in black. (B) Cα-RMSF difference plot for the HNMT simulations. Positive and negative values indicate greater overall fluctuations in the 105T and 105I HNMT proteins, respectively. Secondary structural elements are depicted as for α-helices, and for β-strands, and are colored to match the SAM- and histamine-binding domains shown in Figure 1. * = residue 105.

Figure 3. Snapshots from a MD simulation of 105T HNMT at 37°C

α1 elongates, breaking apart the aromatic cluster within the histamine-binding domain, and exposing E28, Q143 and N283 to the solvent. Hydrophobic interactions between α1 and α11, and salt bridges throughout the domain allow the hydrophobic cluster to reform. This breathing motion may facilitate the entry of charged substrates in a highly hydrophobic environment. Hydrophobic residues within the histamine-binding domain are shown in space-filling representation and colored in green (α1) and red. The catalytic trio (E28, Q143, N283) and the K185-D265 salt bridge are colored by atom and shown in licorice representation.

Figure 4. Distances between core residues within the histamine-binding domains of 105T and 105I HNMT

The contact distances between core residues within the histamine-binding domain (F19-Y147, V173-F243) fluctuate greatly with time, reflecting the periodic breathing motion of HNMT that may facilitate substrate binding. The distance between F19 and F243 is more constant throughout the simulations, as α1 and α11 remain in contact and move together. The figures shows plots of (A) F19 CG (α1) – Y147 OH (α6), (B) F19 CG (α1) – F243 CZ (α11), and (C) V173 CG1 (β5) – F243 CZ (α11) contact distances with time for the 105T (green) and 105I (blue) HNMT simulations at 37°C. (D) Ribbon diagram of HNMT showing the positions of residues F19, Y147, V173 and F243 within the histamine binding domain (red). Residue side-chains are shown in licorice representation and colored by atom.

Figure 5. Snapshots of polymorphic packing taken from the 20 ns structures of the 105T and 105I HNMT MD simulations at 37°C

(A) Packing at the polymorphic site. The larger Ile is more buried, forming side-chain contacts with residues from α3 (L71, S72) and β3 (V111, F113) that are not present in the 105T simulations. (B) The polymorphic packing affects the SAM-binding site. Structural overlay of the polymorphic and SAM binding-sites from the 20ns structures of three independent simulations of 105T and 105I HNMT. The more tightly packed Ile acts as a pivot point between α3, α4 and β3 disrupting the orientation of key SAM-binding residues (E89, P90, Q94, T119). The side chain of the polymorphic residue and its contacts are colored by atom and shown in space-filling and licorice representations, respectively. (C) Distributions of the Cα-RMSDs for residues E89, P90, Q94 and T119 during the last 10ns of all three of the 105T (green) and 105I (blue) HNMT simulations at 37°C. The SAM-binding residues are more mobile in 105I HNMT, existing in a large ensemble of conformations that differ greatly from their respective positions in the starting structure. The distribution for 105T HNMT is narrower, indicating that the active site structure of 105T HNMT is maintained throughout the simulations. The vertical scales are arbitrary.

Figure 6. Translation of polymorphic packing effects to the histamine-binding site

Secondary structures surrounding the polymorphic residue are more susceptible to changes in orientation brought on by altered contacts between L71 and S72 with the larger Ile side-chain. These structural changes are translated to the histamine-binding site via a new contact between α3 (I66) and α2 (E28). (A) Ribbon diagram of HNMT showing the location of residues 105, L71, L72, I66 and E28. Side-chains are shown in space-filling representation and colored by atom. Plots of (B) 105 CG2 – L71 CD2 (α3), and (C) I66 (α3) – E28 (α2, catalytic trio) contact distances for the 105T (green) and 105I (blue) HNMT simulations at 37°C.