Question

Question 2 (a) The partial alignment below shows the highly conserved A, B and C motifs in the nucleotide binding domains of ABC transporters. Some of these amino acid residues they absolutely required to make the proper hydrogen and other are invariant as are bond contacts in the active site. The critical residues in the above sequences are lysine (K) in Walker A; the first aspartate (D) in Walker B; and the SGG sequence in the C- motif. Bearing this in mind, what do these three conserved regions do, collectively? C-motif Walker A Walker B |I I GS 8 GSGKSTF LR I MGP S GS GKS TML N LLGPS G G6 KT TT I L G P S GAGKT T LV G P I I G PN I VGR L V GR L V GR L V G PNG VHLS GGOO RVS ARALAM N Q LS GGQQ R VA1 ARAL AN R ELS GG R R AL GRAIVR R ELS GG QQ R VAL ARAL VK N LS GELS G A GLS G G V LLS GGQ TSLS GGQR S QLS GGOR AVAL ARAL IR V LLFD EPTS AL DP I I LADQ PTGALDS VFMD E P8NL DA LLLLDEPF NL DA L LL LDE MI VMD E ILIFD E I LILDE V L HD E VLILDD ATS MR vRL AAVVLQ LEGR ALMT DV NA T LLR L LR RIA DT L DA RVA N A

Answer 1

The lysine (K) residue in the Walker A motif, together with the main chain NH atoms, are crucial for nucleotide-binding.

It is a glycine-rich loop preceded by a beta strand and followed by an alpha helix; these features are typically part of an α/β domain with four strands sandwiched between two helices on each side. The phosphate groups of the nucleotide are also coordinated to a divalent cation such as a magnesium, calcium, or manganese(II) ion.

Apart from lysine, a feature of the P-loop used in phosphate binding is a compound LRLR nest has the four residues xxGK, whose main chain atoms form a phosphate-sized concavity with the NH groups pointing inwards.

Upon nucleotide hydrolysis the loop does not significantly change the protein conformation, but stays bound to the remaining phosphate groups. Walker motif A-binding has been shown to cause structural changes in the bound nucleotide, along the line of the induced fit model of enzyme binding.

Walker B motif is a motif in most P-loop proteins situated well downstream of the A-motif. The sequence of this motif is [RK]-x(3)-G-x(3)-LhhhD, where R, K, G, L and D denote arginine, lysine, glycine, leucine and aspartic acid residues respectively, x represents any of the 20 standard amino acids and h denotes a hydrophobic amino acid.

This motif was changed to be hhhhDE, where E denotes a glutamate residue. The aspartate and glutamate also form a part of the DEAD/DEAH motifs found in helicases. The aspartate residue co-ordinates magnesium ions, and the glutamate is essential for ATP hydrolysis. There is considerable variability in the sequence of this motif, with the only invariant features being a negatively charged residue following a stretch of bulky, hydrophobic amino acids.

Following the hydrolysis of ADP, a conformational change must occur to separate the ATP-binding cassette. This separation is driven by an electrostatic repulsion by the ADP product that is bound the Walker A motif and the inorganic phosphate product is bound to the C motif.