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Transcript
Module 0220502 Membrane Biogenesis and Transport Lecture 13 ATP-driven Pumps Dale Sanders 2 March 2009 Aims: By the end of the lecture you should understand… • The distinguishing features and physiological importance of P-type ATPases; • The way in which P-ATPase structure is related to mechanisms of ion translocation; • What CPx ATPases are, and how they are related to other P-type ATPases; • The structural attributes and significance of ABC transporters. Reading Lodish et al (2004) Molecular Cell Biol 5th ed, pp.252-7 A good, brief, introduction to P-type ATPases. More detailed accounts: • Toyoshima et al. (2000) Nature 405: 647 • Olesen et al. (2007) Nature 450: 1036 papers on SR Ca2+-ATPase structure • Morth et al. (2007) Nature 450: 1043 paper on Na+,K+-ATPase structure • Solioz & Vulpe (1996) Trend. Biochem. Sci. 21: 237 article on CPx ATPases • Higgins (2007) Nature 446: 749 ABC transporters and multidrug resistance P-Type ATPases: A Widespread Family with Fundamental Physiological Roles UNIFYING FEATURES: • Single large catalytic monomer, 70 – 200 kDa • Inhibition by μM orthovanadate, H2VO4• ATP donates γ- to conserved Asp residue during catalysis • all pump irreversibily • all pump cations What do they do? Some Examples… 1.(Na+/K+) - ATPase of animal cell plasma membranes + – ATP + – 3Na+/2K+/ATP : 3Na+ ADP + Pi – An electrogenic pump 2K+ + • Maintains K+-rich cytosol – essential for protein synthesis etc. - probably originally its primary function • Maintains a Na+-motive force: used to energize coupled transport. • Both Na+ and K+ gradient exploited during electrical signalling. A “housekeeping” enzyme Synergistic stimulation of ATPase activity by Na+ and K+ in animal plasma membranes: ATPase activity Na+ = 40 mM Na+ = 10 mM Na+ = 0 [K] / mM 120 2. Fungal, plant H+-ATPase pH 7 ATP + H ADP + Pi Note: 1H+ /ATP pH 5 or lower • expels excess H+ produced during metabolism: cytosolic pH regulation • maintains PMF: H+ gradient used to energize transport 3. Sarcoplasmic Reticulum Ca2+ - ATPase [also on ER, hence “SERCA” pump] Note: an intracellular membrane myofibrils SR sarcoplasm mM nM Ca2+ 2Ca2+ ADP + Pi 2H+ ATP • Sequesters Ca2+ in relaxed muscle 4. Plasma membrane Ca2+-ATPase Ca2+ ATP nM Ca2+ ADP + Pi mM Ca2+ • Ubiquitous in eukaryotes 2H+ In vivo activity ~ 5% of (Na+/K+)-ATPase Maintains low [Ca2+]cyt in order to… …eliminate Ca2+ toxicity …serve as baseline for amplification during signalling • Ca2+ activation through calmodulin binding domain 5. Gastric mucosal H+/K+-ATPase stomach K+ H+ lumen ATP H+ blood ADP + Pi pH 7.5 pH 1.0 • Unlike other pumps, this is electroneutral, not electrogenic • Acidification of stomach lumen • A target for proton pump inhibitors used to counter diseases associated with stomach acidity e.g. dyspepsia, stomach ulcers Omeprazole, an H/K-ATPase inhibitor 6. E coli K+ uptake system: Kdp-ATPase ATP + K ADP + Pi • A bacterial example • Upregulated at low external [K+] Net cation import Biochemical Properties: The Na+/K+ ATPase • Trypsin cleavage sites are differentially exposed depending on presence of Na+ or K+: ion-dependent conformational changes 100 kDa N C + Na+ + K+ • trypsin cleavage sites The phosphorylated (E-P) intermediate is stabilized by Na+ and ATP in the absence of K+: E-P intermediate is discharged by K+ Mechanism of Transport Results have given rise to the “E1E2” model for catalysis and transport: K+ Na+, ATP IN OUT ADP The “Post-Albers” scheme (K)E1 E1-P(Na) (K)E2 E2-P(Na) Pi K+ Na+ [compare F- & V-type ATPases, where no covalent phosphorylation] H2VO4– competes with Pi for binding; stabilizes transition state For this enzyme, E1 is Na+- and ATP-binding [Ca2+ binding for Ca2+-ATPases] E2 is K+-binding Significance of Conformational States “E1 & E2” conformational states are associated with binding site orientation: ATP hydrolysis drives alternation in binding site orientation In addition there is a change in binding site affinity: allows ion pick-up at low concn on one side allows ion release at high concn on other side These are the two ways in which free energy from ATP hydrolysis is expended. Structure of P-ATPases Hydropathy analysis: cytosolic side N T G T T K G D* E S G D G X N D * the phosphorylated asp residue C only 4% on extracytosolic side SR Ca2+-ATPase at 2.6 Å resolution – Ca2+ binding state (E1) 80 Å (8 nm) A domain contains conserved TGES motif MacLennan & Green (2000) Nature 405: 633 Transport Mechanism of Ca2+ATPase Comparing this structure with that in Ca2+- free (E2) state…… 1. Kinase activity unleashes N domain from P domain 2. A domain associates with N & P domains, exerts downward push on M3/M4, opening luminal pathway for Ca2+ release 3. ATP binding prevents reversal, TGES triggers dephosphorylation 4. Cytoplasmic pathway opens for Ca2+binding Olesen et al. (2007) Nature 450: 1036 CPx – ATPases are P-TYPE ATPases that Transport Heavy Metals Examples: Cd2+ export: in many bacteria, plants Cu+ import: bacterial, human intestine* Cu+ export: toxic levels disposed of in bacteria, yeast, human * Defect Cu deficiency in brain: Menkes and Wilson diseases Structure of CPx ATPases C X X C C X X C C X X C C X X C C X X C C X X C T G E S T G T K D N G D G X N D C X P C Note: N terminal extension CXXC motif found in other metal (eg Hg2+) binding proteins Intramembrane CPx (= CPC, CPH or CPS) motif conserved among all this subclass Absence of extensive transmembrane spans at C terminus Analysing CPx Transporter Function: Complementation of a Yeast Mutant Defective in Cd2+ Tolerance By an Arabidopsis CPx Transporter (AtHMA4) Yeast mutant (ycf1) Wild type Control AtHMA4-expressing Mills et al. (2005) FEBS Lett. 579: 783 HMA4 is a key to tolerating heavy metals in plants (Hanikenne et al. (2008) Nature 453:391) Constitutive overexpression of HMA4 in Arabidopsis halleri facilitates growth on heavy metals. ABC Transporters Ubiquitous: a diverse class, a superfamily, unified by presence of ATP Binding Cassette in 1º structure: GX(S,T)GXGK(S,T)(S,T) Initially discovered as Binding protein-dependent uptake systems that are: Exclusive to Gram -ve bacteria Sensitive to osmotic shock: lose capacity for uptake of small solutes (e.g. histidine, glutamine, arginine) due to release of binding proteins in periplasmic space. In bacteria, binding proteins can mediate interaction between solute and ATP-dependent transport system in inner membrane Solute binding protein ATP Small solute (eg his) Membranebound ATPase component Inner membrane (tight) Outer membrane (permeable to small solutes) System distinct from F-, V-, + P-type 1º pumps ABC Transporters Translocate a Wide Variety of Solutes Some examples: Species System Substrate Direction Streptococcus pneumoniae AmiABCDEF Oligopeptides In E coli HisJQMP Histidine In E coli PstABC Phosphate In Erwinia chrysanthemi PrtD Proteases Out Yeast STE6 a-mating peptide Out Human MDR1 Hydrophobic drugs Out Human CFTR Chloride Human RING 4-11 Peptides Out Into E.R. ABC Transporters Share a Common Domain Structure Binding protein (Gram –ve bacteria only) Tramsmembrane Domain (TMD) Membrane Encoded: 6 t/m spans A C ATP binding 6 t/m spans B D ATP binding Nucleotide-binding Domain (NBD) all on separate genes (esp. prokaryotes) or fused pairwise …. either AB & CD or AC & BD or all one 1 gene (eg MDR1, CFTR) A dimer: 2 x (TMD + NBD) Membrane chamber, open extracellularly Intracellular loops ADP From intracellular side From extracellular side Higgins (2007) Nature 446: 749 Membrane Structure of a Bacterial ABC Multidrug-Transporter (Sav1866 from Staphylococcus aureus) Some Substrates of Multidrug Transporters • Heterocyclic • Lipophilic • Mr < 800 Clinical Implications – and Mechanistic ones too MDRI: “Multidrug Resistance” – “P-glycoprotein” Over-expressed in cells resistant to chemotherapy Responsible for cytosolic clearance of a range of drugs CFTR: “Cystic Fibrosis Transmembrane– conductance Regulator” The CI- channel defective in CF maps to this ABC-type transporter Unusual to have a channel with the structure of a pump! Summary 1. “P-type” ATPases are major cation pumps, performing a variety of functions. 2. They form a phosphorylated intermediate and undergo discrete conformational changes during the catalytic cycle. 3. The conformational changes are associated with (a) binding site reorientation and (b) binding site affinity changes. 4. CPx–ATPases are a distinct sub-class of P-ATPase, and pump heavy metals. 5. ABC transporters pump a wide variety of solutes and are characterised by two distinct Nucleotide-Binding Domains.