Impact of dimerization and N3 binding on molecular dynamics of SARS-CoV and SARS-CoV-2 main proteases

SARS-CoV-2 is a highly contagious and dangerous coronavirus that first appeared in late 2019 causing COVID-19, a pandemic of acute respiratory illnesses that is still a threat to health and the general public safety. We performed deep docking studies of 800 M unique compounds in both the active and allosteric sites of the SARS-COV-2 Main Protease (Mpro) and the 15 M and 13 M virtual hits obtained were further taken for conventional docking and molecular dynamic (MD) studies. The best XP Glide docking scores obtained were −14.242 and −12.059 kcal/mol by CHEMBL591669 and the highest binding affinities were −10.5 kcal/mol (from 444215) and −11.2 kcal/mol (from NPC95421) for active and allosteric sites, respectively. Some hits can bind both sites making them a great area of concern. Re-docking of 8 random allosteric complexes in the active site shows a significant reduction in docking scores with a t-test P value of 2.532 × 10−11 at 95% confidence. Some specific interactions have higher elevations in docking scores. MD studies on 15 complexes show that single-ligand systems are stable as compared to double-ligand systems, and the allosteric binders identified are shown to modulate the active site binding as evidenced by the changes in the interaction patterns and stability of ligands and active site residues. When an allosteric complex was docked to the second monomer to check for homodimer formation, the validated homodimer could not be re-established, further supporting the potential of the identified allosteric binders. These findings could be important in developing novel therapeutics against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

DHFR, dihydrofolate reductase; TS, thymidylate synthase; PRPP, phosphoribopyrophosphate.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.

Insulin-degrading enzyme (IDE) exists primarily as a dimer being unique among the zinc metalloproteases in that it exhibits allosteric kinetics with small synthetic peptide substrates. In addition the IDE reaction rate is increased by small peptides that bind to a distal site within the substrate binding site. We have generated mixed dimers of IDE in which one or both subunits contain mutations that affect activity. The mutation Y609F in the distal part of the substrate binding site of the active subunit blocks allosteric activation regardless of the activity of the other subunit. This effect shows that substrate or small peptide activation occurs through a cis effect. A mixed dimer composed of one wild-type subunit and the other subunit containing a mutation that neither permits substrate binding nor catalysis (H112Q) exhibits the same turnover number per active subunit as wild-type IDE. In contrast, a mixed dimer in which one subunit contains the wild-type sequence and the other contains a mutation that permits substrate binding, but not catalysis (E111F), exhibits a decrease in turnover number. This indicates a negative trans effect of substrate binding at the active site. On the other hand, activation in trans is observed with extended substrates that occupy both the active and distal sites. Comparison of the binding of an amyloid β peptide analog to wild-type IDE and to the Y609F mutant showed no difference in affinity, indicating that Y609 does not play a significant role in substrate binding at the distal site.

MutS, MutL and MutH are the three essential proteins for initiation of methyl-directed DNA mismatch repair to correct mistakes made during DNA replication in Escherichia coli. MutH cleaves a newly synthesized and unmethylated daughter strand 5' to the sequence d(GATC) in a hemi-methylated duplex. Activation of MutH requires the recognition of a DNA mismatch by MutS and MutL. We have crystallized MutH in two space groups and solved the structures at 1.7 and 2.3 A resolution, respectively. The active site of MutH is located at an interface between two subdomains that pivot relative to one another, as revealed by comparison of the crystal structures, and this presumably regulates the nuclease activity. The relative motion of the two subdomains in MutH correlates with the position of a protruding C-terminal helix. This helix appears to act as a molecular lever through which MutS and MutL may communicate the detection of a DNA mismatch and activate MutH. With sequence homology to Sau3AI and structural similarity to PvuII endonuclease, MutH is clearly related to these enzymes by divergent evolution, and this suggests that type II restriction endonucleases evolved from a common ancestor.

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

The metalloenzyme Escherichia coli (E. coli) Alkaline Phosphatase (AP) has a homo-dimeric quaternary structure, which is essential for the enzyme to achieve its catalytic activity, the acceleration of phos- phoester hydrolysis. Employing computational approaches, at differ- ent levels of complexity and sophistication, this thesis aims at under- standing the interplay of structure, dynamics and function of this im- portant enzyme, that is responsible for the supply of vital inorganic phosphate. At low resolution, coarse grained models of the enzyme are con- structed, using an Elastic Network Model (ENM), to explain the in- fluence of the global dynamics, defined by the quaternary structure of the enzyme, on its functionality. Comparative analysis of the col- lective motions, of individual subunits within a homo-dimer of apo and holo enzymes, allows us to interpret the experimental proposal of negative cooperativity. The intrinsic asymmetry of the subunits within the dimer, is already encoded in the enzymes three-dimensional structure, as becomes apparent from the normal mode analysis of the low resolution ENMs, with alternate opening and closing motions present in only one subunit at a time. These results, in conjunction with the analysis of Molecular Dynam- ics (MD) simulations of the atomistic models, at the nanosecond time scale, suggest a mechanical coupling between the correlated motions of individual subunits and their active sites, via the dimer interface. The negative cooperativity of the subunits is further explained by the analysis of multiple, independent MD simulations that reveals subtle differences in the hydrogen bonding network of the subunits and dy- namics of their active site residues, demonstrating a distinct asymmet- ric behaviour, with increased flexibility of one subunit versus rigidity of a second one. Information about structural changes is transmitted between the subunits via the hydrogen bonding network across the in- terface. At least two such communication pathways can be proposed, based on the analysis of MD simulations, both via the interfacial alpha- helix containing residues T559 and T555 that are hydrogen bonded to the residue N416, which is in turn, connected to the residues in the active site. Analysis of the MD simulations confirms that, while the active site of one subunit retains the inorganic phosphate product, as demon- strated by the small changes in the distances between the active site residues, another subunit is letting the product go in order to make the active site available for another turnover of substrate binding. vii Analyses of the MD simulations further suggest that correlated mo- tions of the monomeric subunits of the dimer, as well as the dynamics, and hence the architecture of the active sites, play an important role in the functionality of Alkaline Phosphatase. Although each subunit of the enzyme is equipped with its own catalytic sites, a monomeric AP does not exist in nature and the engineered mutants have significantly reduced activity. Due to the absence of a crystal structure, a model of a monomeric form of the enzyme is constructed based on the crys- tal structure of the apo dimer. In addition, a T59R mutant is built, where interface residues T59 and T559 are substituted by a bulky and charged Argenine residues. Experimental evidence suggests that such a substitution destabilizes the dimeric interface, resulting in a separation into isolated monomers, with reduced structural stability and catalytic activity. Our MD simulation results confirm that the over- all dynamic behaviour of the monomer is different from that of the corresponding dimer and resembles more that of the T59R mutant. Furthermore, comparative analysis based on MD simulations of di- meric and monomeric forms of AP reveals important structural and dynamic features enabling the native dimer to be catalytically func- tional. The stabilisation provided by the interface of the two subunits in the dimeric form of AP is found to be essential for a catalytically competent structure of the active sites. Breaking of the hydrogen bond between residues Y402 and D330, that are located near the active site, as observed in the MD simulations of the monomer, results in the incorrect positioning of the catalytically important, divalent, zinc ion. Understanding the nature of the correlated motions of the subunits within the dimer, and their connection to the enzyme’s activity is an important step in completing our knowledge on structure-dynamics- function relationship of E. coli Alkaline Phosphatase and related en- zymes. Our findings confirm that the structural stability of dimeric AP, provided by the hydrogen bonding network across the interface, is essential for the enzymatic activity. By a combination of different computational approaches, we gained an in-depth understanding of the relationship between the enzymes’ dimeric quaternary structure and its functionality.

Developing allosteric drugs to treat pathogenic diseases can offer a promising alternative to orthosteric drugs that may bind to conserved motifs in human homologs. The allosteric drugs bind to allosteric sites, induce changes in the target protein's active site, and modulate its function with high selectivity, reduced adverse effects, and low toxicity. While identifying allosteric sites is costly and labor-intensive with experimental approaches, computational methods utilizing three-dimensional protein structures offer a cost-effective solution for discovering potential allosteric sites and predicting the effects of ligand binding. This study evaluates the effectiveness of two network models, the residue interaction network (RIN) model and the mixed coarse-grained anisotropic network model (mcgANM), in identifying putative allosteric regions, predicting the structural response of the protein to ligand binding, and elucidating allosteric mechanisms while maintaining computational efficiency. The SARS-CoV-2 main protease (Mpro) is employed as an allosteric protein model due to a rich experimental and computational data available since the COVID-19 pandemic. The findings of the methods are assessed with statistical analysis, all-atom molecular dynamics simulations, and other elastic network models, namely Essential Site Scanning Analysis and Gaussian Network Model using a dataset of 15 ligand-bound and 4 ligand-free structures. RIN predicted the known drug binding sites of Mpro with high statistics, up to 80.0% sensitivity, 89.7% specificity, 29.6% precision, and 89.2% accuracy. RIN suggested an allosteric mechanism of Mpro that facilitates the allosteric communication of the allosteric and active sites through residue fluctuations. RIN was able to decompose the enzyme structure to dynamic domains, showing the organization of structural components to form a functional viral protease. mcgANM suggested the changes in residue fluctuations after ligand binding. The findings underscore the utility of the network models in advancing allosteric drug design.

Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.

An Allosteric Circuit in Caspase-1

Solvent accessibility of βB2-crystallin and local structural changes due to deamidation at the dimer interface

Ribose-5-phosphate isomerase (Rpi), an important enzyme in the pentose phosphate pathway, catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate. Two unrelated isomerases have been identified, RpiA and RpiB, with different structures and active site residues. The reaction catalyzed by both enzymes is thought to proceed via a high energy enediolate intermediate, by analogy to other carbohydrate isomerases. Here we present studies of RpiB from Mycobacterium tuberculosis together with small molecules designed to resemble the enediolate intermediate. The relative affinities of these inhibitors for RpiB have a different pattern than that observed previously for the RpiA from spinach. X-ray structures of RpiB in complex with the inhibitors 4-phospho-d-erythronohydroxamic acid (K(m) 57 microm) and 4-phospho-d-erythronate (K(i) 1.7 mm) refined to resolutions of 2.1 and 2.2 A, respectively, allowed us to assign roles for most active site residues. These results, combined with docking of the substrates in the position of the most effective inhibitor, now allow us to outline the reaction mechanism for RpiBs. Both enzymes have residues that can catalyze opening of the furanose ring of the ribose 5-phosphate and so can improve the efficiency of the reaction. Both enzymes also have an acidic residue that acts as a base in the isomerization step. A lysine residue in RpiAs provides for more efficient stabilization of the intermediate than the corresponding uncharged groups of RpiBs; this same feature lies behind the more efficient binding of RpiA to 4-phospho-d-erythronate.

Structural Basis for Catalytic Activation of a Serine Recombinase