Browsing by Author "Hutter, Michael C."

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca2+ ion pump. We recently,solved the three-dimensional structure of chemically synthesized, unphosphoryloted, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two alpha-helical regions connected by a beta urn- (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) co-workers and modeled into, its E-2 form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other trans membrane helices, as the PLN, binding partner within the membrane and is the only, one of these helices that also permits contact between the N-terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C-terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N-terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen-bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E-2-conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N5,N10‐methylenetetrahydromethanopterin (methylene‐H4MPT) or N5,N10‐methylenetetrahydrofolate (methylene‐H4F) to the respective N5,N10‐methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re‐face‐stereospecific removal of the pro‐R hydrogen atom of the coenzyme's methylene group. The Re‐face stereospecificity is in contrast to the finding that in solution the pro‐S hydrogen atom of methylene‐H4MPT and of methylene‐H4F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene‐H4MPT in solution and when enzyme‐bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N10. The methylene pro‐S C−H bond is anticlinal and the methylene pro‐R C−H bond is synclinal to the lone electron pair of N10. Semiempirical quantum mechanical calculations of heats of formation of methylene‐H4MPT and methylene‐H4F indicate that changing this conformation into an activated one in which the pro‐S C−H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a ΔΔHequation image of +53 kJ mol−1 for methylene‐H4MPT and of +51 kJ mol−1 for methylene‐H4F. This is almost twice the energy required to force the imidazolidine ring in the enzyme‐bound conformation of methylene‐H4MPT (+29 kJ mol−1) or of methylene‐H4F (+35 kJ mol−1) into an activated conformation in which the pro‐R hydrogen atom is antiperiplanar to the lone electron pair of N10. The much lower energy for pro‐R hydrogen activation thus probably predetermines the Re‐face stereospecificity of the four dehydrogenases. Results are also presented explaining why the chemical reduction of methenyl‐H4MPT+ and methenyl‐H4F+ with NaBD4 proceeds Si‐face‐specific, in contrast to the enzyme‐catalysed reaction.