We ran multiple simulations of both 105T and 105I HNMT to examine the effects of the T105I polymorphism on the structure and dynamics of the protein. The root-mean-square deviation of the Cα atoms (Cα-RMSD) between the 105T and 105I HNMT starting structures after energy minimization was 0.4 Å. The overall HNMT structure was maintained throughout all of the simulations, and the polymorphism did not grossly disrupt the protein. However, the structures of both variants expanded and became slightly flattened during the simulations, while their overall solvent-accessible surface area (SASA) increased. This was most likely due to the removal of both the SAH and 2PM substrates (). At 37°C, the SAM- and histamine-binding domains both became more exposed to solvent, and the Cα-RMSD of the SAM-binding domain reached ~3Å in both proteins (). However, the histamine-binding domain becomes slightly more disrupted in the 105T HNMT simulations, reaching a Cα-RMSD of 5.2 ± 0.7 Å compared to 3.7 ± 1.3 Å in the 105I protein.
shows the average Cα-root-mean square fluctuations (Cα-RMSF) about the mean structure for the 37°C simulations, along with the crystallographic B-factors of HNMT (2AOT, (28
)). The fluctuations during the simulations are of the same magnitude and follow a pattern similar to the B-factors. The largest Cα–RMSFs were in the helices and loops of both proteins, especially α1, which makes up one face of the histamine-binding domain, α5 (residues 120-130), and the solvent exposed E276. β-strands positioned within the core of the SAM-binding domain demonstrated the smallest fluctuations (). The Cα-RMSF values differ significantly between the105T and 105I HNMT simulations at several places within the protein structure. Residues on the surface of the histamine-binding domain (β7, α10) fluctuate more in the105I HNMT simulations. 105T HNMT exhibits larger fluctuations for residues 145-163 and 243-248, which contain buried core SAM (I142, M144, Y147) and histamine (Y146, Y147, F243, E246) binding residues. The Cα-RMSD and Cα-RMSF values suggest that the histamine-binding domain of HNMT is quite flexible (, ).
Substrate Binding and the Inherent Flexibility of the Histamine-Binding Domain
The histamine-binding domain has a mixed α/β structure and is comprised of residues from both the amino and carboxy termini of HNMT. The interior of the domain is lined with 14 aromatic residues, providing a very hydrophobic pocket for substrate docking and burial. Three polar residues (E28, Q143, N283) and several water molecules form a hydrogen-bonded network at the base of the pocket (). These residues interact directly with the substrate's charged residues to both orient the substrate and catalyze its N
). In addition, residues C196 and E246 positioned at opposite ends of the pocket are important for substrate binding (28
). Because HNMT is inhibited by a variety of rigid, aromatic compounds that share little structural similarity, it has been hypothesized that the histamine-binding domain exhibits an inherent flexibility that allows tight binding of diverse compounds (18
). More importantly, because the bound substrate or inhibitor is completely buried in the crystal structures, the histamine-binding domain must open up in order for the substrate to enter the active site.
The Cα–RMSD of the histamine-binding domain reached 5.2 Å in simulations of 105T HNMT (). This deviation was due mainly to the flexibility of α1 (, ). Several aromatic residues (F9, Y15, F19, F22) align along one side of α1 to form part of the histamine-binding pocket (). α1 reorients and extends during the simulations, separating the domain into two clusters of aromatic residues (). Throughout the simulations, α1 and α11 remain in contact via multiple salt-bridges and a hydrophobic interaction between F22 and F243, and the two helices to move in concert (). A hydrophobic patch (V173, W179, W183, F190, C196, F243) remains available for substrate binding at the back of the pocket (). The motions of α1 open up the histamine-binding domain, increasing the solvent exposure of the active site by ~ 200 Å2 (). There also is a large increase in the solvent accessibility of residues E28, Q143 and N283 at the base of the active site (, ). Overall, this provides a more accessible site for docking an amphipathic substrate than the initial, tightly packed cluster of hydrophobic residues.
Snapshots from a MD simulation of 105T HNMT at 37°C
Distances between core residues within the histamine-binding domains of 105T and 105I HNMT
Interestingly, α1 appeared to move back towards its starting orientation, in several simulations, resulting in a partial repacking of the histamine-binding domain's hydrophobic core (, ). This movement was facilitated by the close association of helices α1 and α11 (). It is possible that SAM binding affects the motion of α1, as the co-substrate interacts with several residues (M32, M144, Y147) positioned near the histamine-binding domain (). This periodic opening and closing, or breathing, of the active site would expose both a hydrophobic patch and the catalytic residues necessary for substrate-docking and orientation, allowing substrates of various sizes and polarities to bind. The α1-α11 arm could then close off the active site, burying the substrate.
Effects of the T105I Polymorphism
Horton et al. (2001)
reported larger crystallographic B-factors for the polymorphic loop of 105I HNMT than for the 105T protein (18
). However, increased flexibility in the residues immediately surrounding I105 was not observed in the simulations (). In the initial structures of both 105T and 105I HNMT, residue 105 formed backbone hydrogen bonds with residues in α4 (L101, V102) and hydrophobic contacts with residues in α3 (L68), α4 (L101, A103, V102, K104) and the adjacent loop (S106, N107, L108) (). The additional hydrogen bond formed between the hydroxyl group of T105 and the backbone carbonyl of L101 was maintained throughout the 105T simulations.
Snapshots of polymorphic packing taken from the 20 ns structures of the 105T and 105I HNMT MD simulations at 37°C
The overall solvent exposure of the polymorphic site was similar in all simulations (); however, large variations in the solvent accessibility of both I105 and its surroundings occurred at 37°C (). Residue 105 was buried to a greater extent in the 105I protein and the larger Ile formed side-chain contacts with additional residues in α3 (L71, S72) and β3 (V111, F113) that were absent in all of the 105T simulations (, , ). These packing differences suggest that any changes in the orientation of the smaller Thr side-chain are buffered by the additional space present in the polymorphic site and have little effect on the overall protein structure (). In contrast, the more tightly interacting Ile appears to act as a pivot point, affecting the orientation of nearby α3, α4 and β3 through its direct side-chain contacts and transmitting these changes to the SAM –binding site (). Several residues at the distal ends of α4 (E89, P90, Q94) and β3 (T119) interact with the adenosine ring of SAM (). Although some of the contact distances and solvent accessibilities of residues within the SAM-binding site remain similar in both proteins (, , ), many of the SAM-binding residues adjacent to the polymorphic site become much more flexible and disordered in the 105I simulations (, , ). This may explain the increase in the apparent KM
of 105I HNMT for SAM (18
Translation of polymorphic packing effects to the histamine-binding site
In contrast to the effects on the SAM-binding site, the active-site distances and the solvent exposure of residues within the histamine-binding domain are larger and show greater fluctuations in the 105T HNMT simulations than those of the 105I protein (, ). The additional contacts of I105 that disorder the SAM-binding site may play a role in stabilizing the histamine-binding domain. The larger Ile forms side-chain contacts with L68, L71 and S72 in α3 (). The reorientation of α3 in the 105I HNMT simulations allows these residues to interact with E28 (α2), a catalytic residue, through I66 (). Neither the I105-L71 nor the E28-I66 interactions occur in the T105 simulations (). The restricted flexibility of this region in 105I HNMT could possibly impair substrate binding, accounting for the slight increase in its apparent KM
for histamine () (18
Interestingly, catechol O
-methyltransferase (COMT), a fellow member of the SAM-dependent methyltransferase fold family, also has a common polymorphism (V108M) that occupies an almost identical position in the COMT tertiary structure as T105I in HNMT (18
). However, unlike T105I in HNMT, the COMT V108M polymorphism appears to result in decreased protein stability (39
) and to have little effect on substrate binding (40
). MD simulations of the COMT variants showed that the larger Met formed closer side-chain contacts with residues within the polymorphic site, resulting in an increased sensitivity to structural changes in nearby helices and a distortion of the SAM-binding site (38
). These changes were propagated throughout the protein, resulting in an increase in the overall SASA of 108M COMT and destabilization of the protein.