Keithballard/sandbox

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Molecular Playground banner: sHSP, a small but mighty protector against aggregation
Molecular Playground banner: sHSP, a small but mighty protector against aggregation
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<scene name='User:Karan_Hingorani/sandbox_2/Banner_1/1'>E. coli Dihydrofolate Reductase bound to Dihydrofolate and NADP+</scene>
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<scene name='60/609774/Dimer_default/2'>Domain Architecture of sHSP Dimer</scene>
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Dihydrofolate Reductase (DHFR) is a crucial metabolic enzyme whose function is to reduce Dihydrofolate to Tetrahydrofolate, which can then be incorporated into the synthesis of Purines and amino acids. DHFR is classified as an oxidoreductase, which uses NADP+ as the electron acceptor (EC: 1.5.1.3). It is ubiquitously found and is now a popular target for anticancer drugs and antibiotics. <scene name='User:Karan_Hingorani/sandbox_2/Apo_dhfr/5'>Apo-DHFR</scene> free of any of its ligands is displayed here.[http://www.ncbi.nlm.nih.gov/pubmed/2185835?dopt=Abstract]
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Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to diseases of protein misfolding. They comprise a conserved core α-crystallin domain (ACD - red) flanked by an evolutionarily variable N-terminal arm (NTA - green) and semi-conserved C-terminal extension (blue). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that make substrate available to the ATP-dependent chaperones for refolding.
===Structure===
===Structure===
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The monomeric molecular weight of sHSPs range from 12-42 kDa. Many sHSPs form homo-oligomers consisting of 12 to >32 subunits per oligomer. The flexible NTA is disordered and is comprised of 3 small helices connected by random coils. The ACD core domain of the sHSP is an antiparellel β-sandwich containing the dimer interface, which is facilitated by a long loop participating in a strand exchange between partner monomers. The CTD contains an IXI motif (Ile147 and Ile 149), that patches the hydrophobic groove between the β4 and β8 strands in the interacting monomer.
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E.coli DHFR is a small 159 amino acid protein approximately 18kDa. It has an a/b structure with eight central B strands and four helices. The protein can be thought to be made up of two subdomains, divided by the active site cleft. The <scene name='User:Karan_Hingorani/sandbox_2/Ade_loop_1/1'>Adenosine binding loop</scene> which consists of residues 38-88 and the major subdomain comprised of about 100 residues. Three loops can be found in the major subdomain and they make up about 50% of this domain. They are the <scene name='User:Karan_Hingorani/sandbox_2/Met20_loop_1/1'>Met20 loop</scene> (residues 9-24), the <scene name='User:Karan_Hingorani/sandbox_2/Fg_loop_1/1'>F-G loop</scene> (residues 116-132)and the <scene name='User:Karan_Hingorani/sandbox_2/Gh_loop_1/1'>G-H loop</scene> (residues 142-150). The Met20 loop assumes different conformations during catalysis and accomodation of ligands is made possible by the 'hinge bending' motion about Lys 38 and Val 88 of the Adenosine binding domain.[http://www.ncbi.nlm.nih.gov/pubmed/15139807]
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===Function===
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sHSPs are believed to act as ATP independent molecular chaperones that are activated during proteotoxic stress by dissociating into an active form, presumably the sHSP dimer. This form has exposed hydrophobic regions which recognize and bind to hydrophobic patches on denaturing substrate protein. These interactions form a large, soluble, heterogeneous sHSP-substrate complex which can coordinate with ATP independent chaperones to refold substrate, or the degradation machinery for proteolysis.
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===Catalysis===
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DHFR catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate using reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH). This system has been key model to decipher enzyme catalysis and the intermediates of the catalytic cycle have been identified by crystallography. CPMG relaxation NMR experiments have also revealed that intermediates in the catalytic cycle exist in equilibrium with the preceding or following intermediate. Thus the binding of ligands seems to happen via a conformational selection rather than the traditional view of induced fit which is used to explain conformation change on ligand binding.[http://www.sciencemag.org/content/313/5793/1638.short]. <scene name='User:Karan_Hingorani/sandbox_2/Lig_bound_1/1'>Holo DHFR</scene> shows the ligands Dihydrofolate and NADP+ positioned in the active site cleft.[http://www.ncbi.nlm.nih.gov/pubmed/2185835?dopt=Abstract]
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===Drug Target===
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Since DHFR is so critically positioned in the metabolic homeostasis of all organsims it has been the target of choice for anti microbial and anti cancer therapy. Inhibitors of this enzyme are essentially folate mimics, methotrexate which was first designed to inhibit <scene name='User:Karan_Hingorani/sandbox_2/Humandhfr_nad_metho_1/1'>Human DHFR</scene> and used as therapy for cancer and autoimmune disorders. Another folate mimic Trimethoprim was developed as an anti bacterial agent, having much more binding specificity to bacterial DHFR than its mammalian counterpart. Both drugs bind in the active site of the enzyme and are irreversibly bound thus ablating enzyme activity.[http://www.ncbi.nlm.nih.gov/pubmed/3054871] [http://www.ncbi.nlm.nih.gov/pubmed/15681865?dopt=Abstract].
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===3D structures of DHFR===
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[[Dihydrofolate reductase]]
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===See Also===
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The wikipedia link on DHFR is also pretty useful for a general background.[[http://en.wikipedia.org/wiki/Dihydrofolate_reductase]]
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===References===
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1. Bystroff C. et al. Biochemistry 1990
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2. Schnell JR. et al. Annu Rev Biophys Biomol Struct. 2004
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3. Boehr DD. et al. Science 2006
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4. Bystroff C. et al. Biochemistry 1990
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5. Dauber-Osguthorpe P et al. Proteins 1988
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6. Cody V. et al. Acta Crystallogr D Biol Crystallogr. 2005
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Revision as of 15:27, 3 December 2014

Cytosolic CI sHSP from wheat (Ta16.9) 1gme

Drag the structure with the mouse to rotate

One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground.

Molecular Playground banner: sHSP, a small but mighty protector against aggregation

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to diseases of protein misfolding. They comprise a conserved core α-crystallin domain (ACD - red) flanked by an evolutionarily variable N-terminal arm (NTA - green) and semi-conserved C-terminal extension (blue). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that make substrate available to the ATP-dependent chaperones for refolding.

Structure

The monomeric molecular weight of sHSPs range from 12-42 kDa. Many sHSPs form homo-oligomers consisting of 12 to >32 subunits per oligomer. The flexible NTA is disordered and is comprised of 3 small helices connected by random coils. The ACD core domain of the sHSP is an antiparellel β-sandwich containing the dimer interface, which is facilitated by a long loop participating in a strand exchange between partner monomers. The CTD contains an IXI motif (Ile147 and Ile 149), that patches the hydrophobic groove between the β4 and β8 strands in the interacting monomer.

Function

sHSPs are believed to act as ATP independent molecular chaperones that are activated during proteotoxic stress by dissociating into an active form, presumably the sHSP dimer. This form has exposed hydrophobic regions which recognize and bind to hydrophobic patches on denaturing substrate protein. These interactions form a large, soluble, heterogeneous sHSP-substrate complex which can coordinate with ATP independent chaperones to refold substrate, or the degradation machinery for proteolysis.

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Keith Ballard

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