Modelling and expression studies of two novel mutations causing factor V deficiency.

Human coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations, Tyr91Asn and Asp2098Tyr, found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation. Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu(2+) binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.

Comparative analysis of different competitive antagonists interaction with NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor.

The antagonist-bound conformation of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor are modeled using the crystal structure of the DCKA (5,7-dichloro-kynurenic acid)-bound form of the NR1 subunit ligand-binding core (S1S2). Five different competitive NMDA receptor antagonists [(1) DL-AP5; (2) DL-AP7; (3) CGP-37847; (4) CGP 39551; (5) (RS)-CPP] have been docked into both NR2A and NR2B subunits. Experimental studies report NR2A and NR2B subunits having dissimilar interactions and affinities towards the antagonists. However, the molecular mechanism of this difference remains unexplored. The distinctive features in the antagonist's interaction with these two different but closely related (approximately 80% sequence identity at this region) subunits are analyzed from the patterns of their hydrogen bonding. The regions directly involved in the antagonist binding have been classified into seven different interaction sites. Two conserved hydrophilic pockets located at both the S1 and S2 domains are found to be crucial for antagonist binding. The positively charged (Lys) residues present at the second interaction site and the invariant residue (Arg) located at the fourth interaction site are seen to influence ligand binding. The geometry of the binding pockets of NR2A and NR2B subunits have been determined from the distance between the C-alpha atoms in the residues interacting with the ligands. The binding pockets are found to be different for NR2A and NR2B. There are gross dissimilarities in competitive antagonist binding between these two subunits. The binding pocket geometry identified in this study may have the potential for future development of selective antagonists for the NR2A or NR2B subunit.

Structural consequences of D481N/K483Q mutation at glycine binding site of NMDA ionotropic glutamate receptors: a molecular dynamics study.

N-Methyl-D-Aspartate (NMDA) receptors are the ligand gated as well as voltage sensitive ionotropic glutamate receptors, widely distributed in the vertebrate central nervous system and they play critical role in the pathogenesis of schizophrenia. Molecular dynamics simulations have been carried out on high resolution crystal structure of NR1 subunit of NMDA receptor ligand binding core (S1S2) in four different conformations. We have investigated consequence of D481N/K483Q double mutation of NR1 subunit from simulation results of (a) glycine bound form (WG), (b) unbound (closed-apo) form (WOG), (c) a double mutated form (DM), and (d) the antagonist (5,7-dichlorokynuric acid) bound form (DCKA). The MD simulations and simulated annealing for 4ns show a distinct conformation for the double mutated conformation that neither follows the antagonist nor apo conformation. There are two distinct sites, loop1 and loop2 where the double mutated structure in its glycine bound form shows significant RMSD deviations as compared to the wild-type. The interactions of glycine with the receptor remain theoretically unchanged in the double mutated structure and there is no detachment of S1S2 domains. The results suggest that separation of S1 and S2 domains may not be essential for channel inactivation. Therefore, it is hypothesized that hypoactivation of NMDA receptor channels may arise out of the conformational changes at non-conserved Loop1 and Loop2 regions observed in the mutated structure. The Loop1 and Loop2 regions responsible for inter-subunit interactions in a functional NMDA receptor, may therefore, render the ligand bound form defunct. This may account for behavioral anomalies due to receptor inactivation seen in grin1 mutated mice.

Evolutionary trace analysis of ionotropic glutamate receptor sequences and modeling the interactions of agonists with different NMDA receptor subunits.

The ionotropic N-methyl- d-aspartate (NMDA) receptor is of importance in neuronal development, functioning, and degeneration in the mammalian central nervous system. The functional NMDA receptor is a heterotetramer comprising two NR1 and two NR2 or NR3 subunits. We have carried out evolutionary trace (ET) analysis of forty ionotropic glutamate receptor (IGRs) sequences to identify and characterize the residues forming the binding socket. We have also modeled the ligand binding core (S1S2) of NMDA receptor subunits using the recently available crystal structure of NR1 subunit ligand binding core which shares approximately 40% homology with other NMDA receptor subunits. A short molecular dynamics simulation of the glycine-bound form of wild-type and double-mutated (D481N; K483Q) NR1 subunit structure shows considerable RMSD at the hinge region of S1S2 segment, where pore forming transmembrane helices are located in the native receptor. It is suggested that the disruption of domain closure could affect ion-channel activation and thereby lead to perturbations in normal animal behavior. In conclusion, we identified the amino acids that form the ligand-binding pocket in many ionotropic glutamate receptors and studied their hydrogen bonded and nonbonded interaction patterns. Finally, the disruption in the S1S2 domain conformation (of NR1 subunit- crystal structure) has been studied with a short molecular dynamics simulation and correlated with some experimental observations. [figure: see text]. The figure shows the binding mechanism of glutamate with NR2B subunit of the NMDA receptor. Glutamate is shown in cpk, hydrogen bonds in dotted lines and amino acids in blue. The amino acids shown here are within a 4-A radius of the ligand (glutamate).