Week 11 Hitchcock Lecture 5

Question 1

What are ABC transporters?

• Why are ABC transporters common for nutrient scavenging?
• What is the general structure of ABC transporters?
• What is the mechanism of ABC transporters?

Answer 1

ATP-binding cassette (ABC) transporters are a family of transporters that use free energy of ATP binding and hydrolysis to move their substrates across membranes

• typically have very high substrate affinity and high accumulation rates, which means they can concentrate substates inside cells to very high levels using energy from ATP
• 2 Integral membrane (IM) transporter subunits;
• -Two subunits form a homodimer/heterodimer with 12 amphipathic TMHs.
• -Translocate substrate through the membrane.
• 2 Cytoplasmic ATP-binding cassette (ABC) subunits
• -Highly conserved sequence
• -ABC signature, Walker A and Walker B motifs bind ATP
• -Hydrolyse 2 mol ATP per mol of substrate transported
• -Adopt open and closed conformations - short coupling helices transmit conformational changes to the IM domains
• Periplasmic substrate binding protein (SBP)(uptake systems only)
• -Bind substate with high affinity and deliver it to IM subunits
• -Substrate binding pocket between two domains joined by a central hinge - large conformational change between open and closed states
• -May (always in Gram-positives) be lipid anchored
• Mechanism;
• -The transport cycle starts with an inward-facing conformation
• -Substrate-loaded SBP approaches periplasmic side of the transporter
• -ATP binds to ABC subunits, which close and push the coupling helices toward each other and convert the IM domains into an outward-facing conformation
• -Tunnel for substrate from SBP to binding pocket in IM subunits halfway across the membrane
• -ATP is hydrolysed causing the ABC dimer to open - this pulls the coupling helices outward and triggers conversion of the IM subunits to the inward-facing conformation
• -Substrate released into the cytoplasm and system resets

Question 2

What is the general function of Ion-translocating P-type ATPases?

• What is the general structure of Ion-translocating P-type ATPases?
• Explain the function of the 3 cytoplasmic domains

Answer 2

Use energy from ATP hydrolysis to pump cations across the cell membrane.

• 70-150 kDa proteins with four conserved domains; a 6-10 TMH M domain binds and transports the ions and three cytoplasmic domains (P, N and A).
• Domains;
• -Named P-type as they contain a key aspartate residue (found in a characteristic DKTGT motif) in the cytoplasmic phosphorylation (P) domain which is phosphorylated during the transport cycle
• -The nucleotide binding domain (N) domain delivers ATP to the P domain to phosphorylate the Asp residue
• -The actuator domain (A) domain de-phosphorylates the P domain and transduces the energy from ATP hydrolysis in cytoplasmic domains to the transport of ions in the M domain
• -May interact with additional proteins such as chaperones for ion delivery or regulatory subunits

Question 3

What is CopA?

• What is the primary role of CopA?
• What is the general structure of CopA?
• What are CopZ and CueO?

Answer 3

a copper(I)-exporting bacterial P-type ATPase

• the role of CopA is to pump Cu(I) from the cytoplasm to the periplasm when copper levels are too high
• 8 TMHs in the M domain, the typical N, P and A cytoplasmic domains, additional fused N-terminal metal binding domains
• CopZ
• -is a metallochaperone that binds Cu(I) in the cytoplasm and delivers it to CopA
• -CueO is a multi-copper oxidise that oxidises Cu(I) to Cu(II) in the periplasm

Question 4

What is the general mechanism of Ion-translocating P-type ATPases?

Answer 4

Mechanism;

• High affinity ion binding to M domain binding sites requires ATP is bound to the N domain (periplasmic access blocked)
• ATP hydrolysis at the N domain phosphorylates Asp residue on P domain causing conformational changes
• Ion now has lower affinity and is released to the periplasmic side of the membrane (cytoplasmic access blocked)
• ADP is released, the P domain is dephosphorylated by A domain and ATP binds to the N domain, resetting the system

Question 5

What is the phosphotransferase system (PTS)?

• What does PTS do?
• What is the general idea of PTS’s?
• How do PTSs work?
• What are the other derivatives of the PTS structure?

Answer 5

Phosphoenolpyruvate (PEP) group translocation
-Bacterial primary active transport system for sugar uptake (e.g. glucose, mannose, fructose)
-The sugar is phosphorylated into a sugar-phosphate during the transport process (e.g. glucose-6-P). Phosphorylated sugar ‘trapped’ in the cytoplasm and can directly enter e.g. glycolysis or the pentose phosphate pathway. Driven by hydrolysis of phosphoenolpyruvate (PEP) – an intermediate in glucose catabolism
-Mechanism;
–have this phosphotransferase system where first the phosphate group on phosphoenolpyruvate is transferred to enzyme I, then onto the phosphoryl carrier protein called HPr, which has a conserved His residue that accepts the phosphoryl group
–HPr transfers the phosphoryl group onto enzyme EIIA which then passes it onto a cysteine residue in the enzyme EIIB domain, which is fused to the integral membrane sugar permease, called IIC
–As the glucose is transported by IIC, it is phosphorylated by EIIB forming, glucose-6-phosphate
–Enzyme I and HPr phosphocarrier protein are non
specific components and are involved in phosphotransferase systems for uptake of many different sugars, where as sugar specificity imparted by the enzyme II proteins/subunits
-Include;
–enzyme IIA can be fused to the IIB and IIC domains in a single membrane bound protein
–Or IIA and IIB can be fused as a single two-domain soluble protein which interacts with the permease at the membrane

Question 6

What are Decarboxylation driven transporters?

• How does it work?
• What is its structure?

Answer 6

use the free energy of decarboxylation of organic acids to drive active transport of Na+ ions out of the cell, generating a sodium motive force

• Mechanism;
• -Citrate is taken up by Na+ dependent carrier and converted to acetate and oxaloacetate
• -Oxaloacetate decarboxylated to pyruvate – free energy used to pump Na+across membrane
• -PFL generates acetyl-CoA and formate, which are metabolised as in E. coli
• -H2 produced by FHL used to generate NADH
• Structure:
• -a subunit – peripheral membrane protein facing cytoplasm. Contains carboxyltransferase domain and covalently binds biotin cofactor
• -β subunit – 9 TMH integral membrane protein. Contains biotin decarboxylation site and Na+ ion channels
• -γ subunit – small subunit, contains polyhistidine ligands for binding a catalytically important Zn2+ ions. Anchored by single THM