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FRCPath Self help course 2009 10 th December 2009 Protein motifs

FRCPath Self help course 2009 10 th December 2009 Protein motifs. Nucleotide Binding Domains Stacey Sandell. Introduction. Adenine and guanine nucleoside triphosphate (ATP and GTP) have distinct biological roles.

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FRCPath Self help course 2009 10 th December 2009 Protein motifs

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  1. FRCPath Self help course 200910th December 2009Protein motifs Nucleotide Binding Domains Stacey Sandell

  2. Introduction • Adenine and guanine nucleoside triphosphate (ATP and GTP) have distinct biological roles. • Hydrolysis of ATP provides energy that drives metabolic reactions by enzymes and movement by motor proteins. • Phosphorylation reactions consume ATP. • GTP hydrolysis seems to be used mostly for regulation by guanine nucleotide binding proteins (GNBPs) • GNBPs are molecular switches, cycling between off and on states. They control processes ranging from cell growth and differentiation to vesicular and nuclear transport

  3. GNBPs • The guanine nucleotide binding domain (GNBD) or G domain carries out nucleotide binding and hydrolysis • It contains 4-5 consensus sequences which are lined up around the nucleotide binding site. • N/TKXD motif • GXXXXGKS/T (P loop) The diagram of the ‘universal switch mechanism’ Release of the gamma-phosphate after GTP hydrolysis allows the switch regions to relax into a different conformation.

  4. Nucleotide binding domains of ATPases • NBDs have been shown to dimerise, bind ATP and hydrolyze ATP. • It was initially thought that the ATP hydrolysis provided the energy input but more recent studies suggest that ATP binding rather than ATP hydrolysis provides this energy. • In 1998 the crystal structure of a bacterial NBD was solved. Many human ATPases were discovered based on looking for regions of homology. • ATPases are split into two main groups. • ATP-binding cassette transporters (ABC transporters). • P-type ATPases.

  5. ATP Binding Cassette Transporters • Large Transmembrane protein family that utilise the energy of ATP hydrolysis to drive the transport of various molecules across the cell membrane. • Common features of all ABC transporters is that they consist of 2 distinct domains: • Transmembrane domain (TMD) • Nucleotide binding domain (NBD) • The structural architecture of these proteins consists minimally of 2 TMDs and 2 NBDs. • TMD consists of alpha helices embedded in the membrane bilayer. It recognises a variety of substrates and undergoes conformational changes to transport the substrate across the membrane.

  6. ATP Binding Cassette Transporters • The NBD is located in the cytoplasm and has a highly conserved sequence. In most exporters the N-terminal transmembrane domain and C-terminal NBD are fused and arranged as TMD-NBD-TMD-NBD.

  7. Structure of ABC transporter NBDs • NBDs split into 2 domains • Catalytic core domain • a-helical subdomain • Catalytic core domain • Typically consists of 2 b sheets and 6 a helices. • Walker A motif or P loop (GXXGXGKS/T). • Walker B motif (ffffD) f = hydrophobic residue. • Helical domain • ¾ helices • ABC signature motif (LSGGQ) • Other important regions • Q-loop or g-phosphate switch • Conserved glutamine residue that connects the TMD and ABC • H motif or switch region • Conserved histadine residue, important in the interaction between ABC domain and ATP.

  8. Structure of the NBD of ABC transporters with bound nucleotide

  9. ATP hydrolysis Dimer formation of the ABC domains of transporters require ATP binding. 2 molecules of ATP are positioned at the interface of the dimer, sandwiched between the Walker A motif of one and the LSGGQ motif of the other. The motifs stabilise the ATP binding. The enzymatic hydrolysis of ATP requires proper binding of the phosphates and positioning of the g-phosphate to the attacking water. The precise mechanism is still controversial. The ATP switch model: Two conformations of NBDs: 1. Formation of closed dimer upon two ATP molecules binding. 2. Open dimer facilitated by ATP hydrolysis and release of inorganic phosphate and ADP. This results in substrate tranlocation.

  10. ATP switch model (of an exporter). ATP switch model (of an exporter).

  11. ABC transporters and disease Genetics variability in the ABC transporters is a cause or contributor to a wide variety of human disorders. These include cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anaemia and drug response phenotypes. ABC transporters have gained extensive attention because they contribute to the resistance of cells to antibiotics and anticancer agents, by pumping drugs out of the cell. MDR1/ABCB1 or P-glycoprotein pumps tumour suppression drugs out of the cell. Many other ABC proteins from the different subfamilies also contribute to multi-drug resistance.

  12. CFTR/ABCC7 Most Eukaryotic ABC transporters are effluxers but some are not directly involved in transporting substrates. CFTR uses ATP hydrolysis to regulate the opening an closing of a chloride ion channel. It has the architecture MSD1-NBD1-R-MSD2-NBD2. The NBDs of CFTR are structurally asymmetric NBD1 has an ABC motif and a Walker A motif but lacks conservation of the Walker B motif and the H loop. NBD2 has sequence conservation at Walker A, Walker B and the switch motifs but lacks conservation at the ABC motif. It is predicted that they act in a head to tail orientation with one catalytic site. 1.ATP binding to both sites promotes dimerisation and conformational changes which result in channel opening. 2.Subsequent hydrolysis of ATP at one of the binding sites proceeds channel closing. 3.The channel is also controlled by phosphorylation of the R domain.

  13. Intact CFTR protein forms a chloride-permeable channel in the outer membrane of many cells. The precise structure has yet to be determined, but movement of chloride through the pore is known to be regulated by three cytoplasmic domains of the protein. Passage is allowed only when the two nucleotide-binding domains dock and cleave adenosine triphosphate (ATP) and when the regulatory domain becomes studded with phosphate groups.

  14. Mutations in CFTR DF508 is the most common CFTR mutation in most populations, it is most likely to exert it’s effect by abnormal protein folding than affect ATP binding directly. Studies have shown that the mutation impairs normal protein maturation and trafficking to the plasma membrane. The CF causing mutation G551D situated in the NBD1 signature motif LSGGQ has been shown to reduce ATP binding and hydrolysis.

  15. ABCR/ABCA4 Mutations have been reported to cause a spectrum of autosomal recessively inherited retinopathies including Stargardt disease, cone rod dystrophy and retinitis pigmentosa. Individuals that are heterozygous for ABCR mutations may be predisposed to develop the multifactorial disorder age-related macular degeneration. The gene encodes a photoreceptor-specific ABC transporter of retinaldehyde or a related derivative essential to the vision cycle. Defects in ABCA4 lead to an accumulation of retinal derivatives in the epithelium behind the retina. A proposed model is that the retinal disease severity is correlated inversely with residual ABCA4 activity. Complete loss = retinitis pigmentosa.

  16. P-type or E1/E2 ATPases Large group of evolutionarily related ion pumps. The ATP-binding domain of these proteins was shown to consist of two domains: Phosphorylation domain (P domain) containing the DKTG sequence. Nucleotide binding domain (N domain) which contributes to ATP co-ordination. P-type ATPases are divided into 5 subfamilies based on their ion specificity and structural characteristics. The residues strictly conserved among all P-type ATPases are almost exclusively limited to the P domain and Actuator domain (the most important domains for catalysis). The nucleotide binding domains are equally important but not conserved, this suggests co-ordination of ATP by different P-type ATPase subfamilies could be quite different.

  17. ATP7B P-type ATPase

  18. ATP7B / Wilson Disease Protein (WNDP) The Wilson Disease Protein (WNDP) is a key regulator of copper homeostasis in a number of tissues, particularly liver, brain and kidneys. WNDP transports copper from the cytosol across cell membranes, using the energy of ATP hydrolysis. WNDP’s normal function is to deliver copper to enzymes within the secretory pathway and is essential for copper secretion when copper concentrations are elevated. Mutations in WNDP lead to a marked accumulation of copper in the cytosol and a severe hepatoneurological disorder known as Wilson disease.

  19. References Lewis et al (2004) The EMBO Journal 23:282-293. Stratford et al (2007) Biochem.J. 401:581-586. deCarvalho et al (2002) The Journal of Biological Chemistry277;39:35896-35905. Lewis et al (2005) The Journal of Biological Chemistry280;2:1346-1353. Shroyer (2001) Human Molecular Genetics10;23:2671-2678. Morgan (2004) The Journal of Biological Chemistry279;35:36363-36371.

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