S-adenosylhomocysteine hydrolase (SAHH), a cellular enzyme that plays a key role in methylation reactions including those required for maturation of viral mRNA, is an important drug target in the discovery of antiviral agents. While targeting the active site is a straightforward strategy of enzyme inhibition, evidences of allosteric modulation of active site in many enzymes underscore the molecular origin of signal transduction. Information of co-evolving sequences in SAHH family and the key residues for functional dynamics that can be identified using native topology of the enzyme provide glimpses into how the allosteric signaling network, dispersed over the molecular structure, coordinates intra- and inter-subunit conformational dynamics. To study the link between the allosteric communication and functional dynamics of SAHHs, we performed Brownian dynamics simulations by building a coarse-grained model based on the holo and ligand-bound structures. The simulations of ligand-induced transition revealed that the signal of intra-subunit closure dynamics is transmitted to form inter-subunit contacts, which in turn invoke a precise alignment of active site, followed by the dimer-dimer rotation that compacts the whole tetrameric structure. Further analyses of SAHH dynamics associated with ligand binding provided evidence of both induced fit and population shift mechanisms, and also showed that the transition state ensemble is akin to the ligand-bound state. Besides the formation of enzyme-ligand contacts at the active site, the allosteric couplings from the residues distal to the active site is vital to the enzymatic function.
S -adenosylhomocysteine (SAH) hydrolase catalyzes the hydrolytic cleavage of SAH to adenosine and L-homocysteine. Inhibition of this enzyme causes the accumulation of SAH, and consequently suppresses S -adenosyl-L-methionine dependent transmethylation via a feedback inhibition mechanism. Since the methylation at the 5'-terminus of mRNA is crucial for the viral replication, SAHH is a promising target for the discovery of broad spectrum antiviral agents [1].
Developing therapeutic agents that can directly bind and regulate the active site of a biological target has been a dominant pharmacological strategy [2,3]. For SAHH, various adenosine analogues, including carbocyclic adenosine, neplanocin A, 3-deazaneplanocin A [1], and fluoroneplanocin A (F-NpcA), are the recently developed inhibitors that directly target the active site [4]. However, conformational flexibility of enzyme structures gleaned in x-ray, NMR experiments and the presence of allosteric site revealed in mutational studies highlight the allosteric couplings of residues distal to the active site as an another important principle in drug design strategy [5][6][7]. Although the formation of specific enzyme-substrate contacts in the active site is required for the catalytic activity, allosteric orchestration among residues, dispersed over the molecular architecture, is also essential to regulate conformational fluctuations, so as to assist a precise positioning of catalytic elements in the active site [8]. SAHH, an enzyme consisting of chemically identical four subunits, each of which undergoes the open-to-closed (O→C) transition in response to substrate binding, is an interesting system to study the link between enzymatic function and allosteric dynamics beyond monomeric enzyme [9][10][11][12][13][14][15].
Structure, dynamics and catalytic function are the three main themes in understanding enzymes [6,9,[16][17][18][19][20]. For the past decades, much effort has been made to decode the link between the allosteric signaling of enzymes, conformational dynamics, and their function by using both theories [10,14,[21][22][23][24] and experiments [13,20,[25][26][27] , and has recently been extended to the studies of molecular motors [28][29][30]. To gain microscopic understanding to the allostery in SAHH and its implication to the catalysis, we employed multifaceted computational approaches:
(i) Statistical coupling analysis (SCA) [21,31,32] was used to reveal networks of co-evolving amino acid residues in the SAHH family. (ii) Structural perturbation method (SPM) adapting normal mode analysis (NMA) identified the network of hot spot residues associated with the functional motion of SAHH [23]. (iii) While the SCA and SPM provide hints as to the corre-lation between allosteric signaling network and conformational dynamics, it is difficult to gain from these two static analyses further insights into a large scale conformational change, such as a shifting of statistical ensemble and heterogeneity in the transition routes. To this end, we performed Brownian dynamics (BD) simulations of the O→C transition of tetrameric SAHH in response to ligand binding by using a structure-based coarse-grained model [10,12,30,[33][34][35].
Among the various stages of the SAHH enzymatic cycle that involve multiple catalytic processes [36,37], which occur on time scales of ∼ (10 -100) ms, main focus of our simulation is on the fast (∼ (1 -100) µs) conformational dynamics of SAHH before, after as well as in the process of substrate binding. Despite the large time scale gap between catalysis and conformational dynamics associated with ligand binding, it is important to understand the conformational fluctuations and dynamics since they lie at the core of allosteric regulation of catalytic power in enzymes. In this paper, by analyzing the dynamics resulting from our simulation, we highlight the link between the functional dynamics and allosteric signaling implicated in the SCA and SPM.
SAHH structure: A subunit structure, consisting of 432 residues, retains catalytic, cofactor-binding and C-terminal domains. An alpha helix (αA1) and loops (residues 182-196, 352-354) join the catalytic domain with the cofactor-binding domain, forming a hinge region (Fig. 1A left, see also Figure S1). The interaction between C-terminal domain of a subunit and cofactor-binding domain of the partner subunit (Fig. 1A right) is employed to assemble two dimer complexes (AB and CD) into the tetramer. A negatively charged central core channel constitutes the interface between A and B (or C and D) (Fig. 1B). A further assembly between the two dimers (AB and CD), whose interface exhibits electrostatic complimentarity, forges an oblate-like tetrameric structure (Fig. 1B). Depending on the presence of ligand in the binding cleft between catalytic and cofactor-binding domains, SAHHs select either open or closed conformation whose root-mean-square deviation (RMSD, ∆ OC ) is 4.2 Å. The difference of the two s
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