The Structural Basis of Calcium Dependent Inactivation of the Transient Receptor Potential Vanilloid 5 Channel.

The Transient Receptor Potential Vanilloid Channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+-binding protein Calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterisation of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while conferring conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with, and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate et al. (Biochemistry, 2018, in press)

NMR Structures of Apo L. casei Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

bS Supporting Information The enzyme dihydrofolate reductase (DHFR; 5,6,7,8-tetra-hydrofolate:NADPH oxidoreductase, EC 1.5.1.3) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-folate (THF) using NADPH as coenzyme.1 Since THF and its metabolites are precursors of purine and pyrimidine bases, the normal functioning of this enzyme is essential for proliferating cells. This makes DHFR an excellent target for antifolate drugs such as methotrexate (anticancer), pyrimethamine (antimalarial), and trimethoprim (antibacterial). Such agents act by inhibiting the enzyme in parasitic or malignant cells.1,2 The cooperative binding of ligands to DHFR plays an important role not only in the enzyme catalytic cycle (negative cooperativity in THF/ NADPH binding)3 but also in enzyme inhibition (positive cooperativity in antifolate/NADPH binding).4 The effects of positive cooperative binding in controlling enzyme inhibition ar

NMR Structures of Apo <i>L. casei</i> Dihydrofolate Reductase and Its Complexes with Trimethoprim and NADPH: Contributions to Positive Cooperative Binding from Ligand-Induced Refolding, Conformational Changes, and Interligand Hydrophobic Interactions

In order to examine the origins of the large positive cooperativity (Δ<i>G</i>₀^coop = −2.9 kcal mol⁻¹) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of <i>L. casei</i> apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A−B loop region and part of helix B (residues 15−31) which could not be directly detected for <i>L. casei</i> apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to −0.95 kcals mol⁻¹ if 80% of A−B loop requires refolding). Comparisons of Cα−Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to −1.4 kcal mol⁻¹)