Methotrexate: Difference between revisions

<StructureSection load='2w3m' size='450' side='right' scene='42/420816/Cv/2' caption=''>

The nature of this binding has a 1000 fold increase in affinity relative to the natural folate affinity of DHFR , producing a practically irreversible inhibition of <scene name='Sandbox_58/N_to_c/1'>DHFR</scene> activity, (blue = N-terminal, red C-terminal).  Methotrexate is a competitive inhibitor that can bind to and inhibit the <scene name='Sandbox_58/Dhf_reductase/1'>DHRF active site</scene>, residues displayed in red, and the flexible Met20 loop surrounding the active site displayed in blue. Specifically, methotrexate is able to competitively interact with the <scene name='Sandbox_58/Active_site_mxt/2'>active site </scene> residues of DHFR, specifically Asp27, Phe31, Arg57, and Tyr100, with associations with the Asn18, Leu28, and Ile50 residues. The active site is buried within the enzyme as is depicted by the <scene name='Sandbox_58/Solvent_accessable_surface/1'>solvent accessable surface</scene> shown in orange at the entrance to the active site. The  

<scene name='Sandbox_58/Relative_temperature/1'>relative temperature</scene> are color depictions of each atom in regards to mobility or position uncertainty relative to the molecule, with increasing mobility as the color scheme goes from blue to red.  The interactions of the rest of the protein are depicted through the <scene name='Sandbox_58/H_bonds/1'>hydrogen bonds</scene> displayed in red<ref>Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K (July 1977). "Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate". Science 197 (4302): 452–455.</ref>.  

The features of DHFR ligand binding, specifically to methotrexate can be observed and analyzed through various molecular docking and mutation experiments. The a structurally engineered variant of the <scene name='Sandbox_58/Arg_31-35_norm/1'>native human DHFR</scene> altered the F 31 residue of the protein to R, and the Q 35 residue of the protein to E in an attempt to explore the specifics of the methotrexate affinity for DHFR active site residues, resulting in varied active site residues from phenylalanine and glutamine to <scene name='Sandbox_58/Arg_31_glutamine_35/1'>arginine and glutamate</scene>.  This mutated enzyme featured a 650x decrease in affinity for the ligand, methotrexate, but retained an amount of methotrexate interaction similar to the enzyme in its native state with native substrates.  Crystal structure analysis revealed that the lack of cooperative action and presence of residue disorder lead to the significant decrease in methotrexate activity with the resulting <scene name='Sandbox_58/3eig_active_site/1'>active site</scene>.  The arginine residue at place 31, was specifically observed in numerous conformations, a characteristic unique to the mutated enzyme, and the probable cause of the loss of polar contacts and binding affinity between methotrexate and DHFR.  A loss of van der Waal forces due to the conformations of the side chains along with an unfavorable placement of Glu-35 causing an “unfavorable electrostatic contact” with methotrexate’s “glutamate portion.”  Interestingly this variant was found to display a greater decrease in methotrexate affinity than the decrease in affinity of Dihydrofolate, found to be 9x, evident of catalytic efficiency retention which hold many drug binding resistance implications<ref>Volpato, J., Yachnin, B., & Blanchet, J. (2009). Multiple conformers in active site of human dihydrofolate reductase F31R/Q35E double mutant suggest structural basis for methotrexate resistance.. Journal Biol. Chem., 284, 20079-20089. </ref>.