User:Eran Hodis/Sandbox3
Your Heading Here (maybe something like 'Structure')
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Structural Features
General StructureThere are two distinct classes of HMGRs, class I, which is only found in eukaryotes and are membrane bound and class II, which is found in prokaryotes and are soluble. [1] HMGR contains 8 transmembrane domains that have yet to be successfully crystallized, which anchor the protein to the membrane of the endoplasmic reticulum. [2] The catalytic portion of human HMGR forms a tetramer, with the individual monomers winding around each other. [2] Within the tetramer, the monomers are arranged into two dimers, each of which contains two active sites which are formed by residues form both monomers. Each monomer contains three domains , the N-domain, the L-Domain, and the S-Domain. The L-domain is unique to HMGRs while the S-domain, which forms the binding site for NADP, resembles that of ferredoxin. The S and L domains are connected by a “cis-loop” which is essential for the HMG-binding site. [2] Salt bridges between residues R641 and E782 as well as hydrogen bonds between E700 and E700 on neighboring monomers compliment the largely hydrophobic dimer-dimer interface. [2]
Substrate Binding & Catalytic Mechanism The HMG-CoA and NADPH molecules make numerous contacts with the L and S domains in forming the four active sites. The CoA is located in a near the enzyme surface, with the pantothenic acid moiety extending into the interior of the protein. Tyrosine 479 forms a hydrophobic lid over the CoA adenine base, shielding the extended binding pocket from solution. The NADPH binding site is formed primarily by the S-domain with a loop region playing a critical role in binding. [2] The HMG binding pocket is the site of catalysis in HMGR. The“cis-loop” that bends over the top of HMG is a critical structural element of this binding site. Residues E559 and D767 and are positioned in the active site as is K691 which is only 2.7 angstroms from the HMG O2 carbonyl oxygen. It is this K691 that likely stabilizes the negatively charged oxygen of the first mevaldyl-CoA intermediate. [2] The mevaldyl CoA intermediate is subsequently converted to Mavaldehyde with added stabilization from H866, which is within hydrogen bonding distance of the thiol group. It is then believed that the close proximity of E559 and D767 increases the pKA of E559, allowing it to be a proton donor for the reduction of mevaldehyde into mevalonate. [2]
Regulation of HMG-CoA ReductaseAs stated before, HMGR is among the most highly regulated enzymes in the human body.[3] Regulation of HMG-CoA reductase occurs at 4 different levels of feedback regulation. First by regulation of transcription of the reductase gene, which is activated by sterol regulatory element binding protein, a protein that binds to the promoter of the HMGR gene when cholesterol levels fall. The second level of regulation is at the translation of the HMGR mRNA, which is inhibited by Farnesol, a derivative of the mevalonate pathway.[3] The third level of HMGR regulation involves the degradation of intact reductase. As the level of sterols increases, HMGR reductase becomes more susceptible to ER-associated degradation. Helices 2-6 of the HMGR transmembrane domain, called the sterol-sensing domain, sense the increased levels of sterols. Degradation of HMGR is initiated when a membrane-bound cysteine protease cleaves residues within the transmembrane region of HMGR. Additionally, as sterol levels increase, the helices can expose Lysine 248 which can be ubiquitinated and subsequently trigger proteolytic degradation.[2][4] A final level of HMGR regulation is achieved by inhibition via phosphorylation of Serine 872. HMGR is phosphorylated and inactivated by an AMP-activated protein kinase, when the energy charge of the cell is low and AMP concentrations are high. Since Serine 872 is in the vicinity of the pohsphate of NADP, phosphorylation likely results in a decreased affinity for NADPH, halting the formation of Mevaldyl-CoA from HMG-CoA.[5]
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