The structure of eukaryotic ADPGK

The overall fold of M. musculus ADPGK is remarkably similar to the archaeal ADPGKs (see section 3.4.5). However, the solved structure comprises residues 51 to 496, excluding the putative N-terminal signal peptide. The region between arginine residue 74 to serine residue 99 has been described to be an amphipathic, membrane associated helix in a recent study (Kamiński et al. 2012). However, this is not supported by the solved structure of rmADPGKΔ51. The region in question (between residue position markers 73 and 99 in Figure 77, coloured blue) is an internal strand of the central β-sheet, transitioning into a hinge-region β-strand and ultimately a short helix part of the small lid domain (blue α-helix preceding marker 99 in Figure 77). This helix in the small domain packs against the hydrophobic core of the lid. A membrane association of this part of the protein is therefore unlikely without a major structural rearrangement.

Interestingly, a disulphide bond was found in the structure of mouse ADPGK. Both cysteine residues are conserved in ADPGK of other eukaryotes, but not in archaea, as can be seen in the protein sequence alignment in Figure 80. In a BLAST search with

the H. sapiens ADPGK sequence an uncharacterized protein from Tetrahymena

thermophila strain SB210 (accession code I7M6W2) was found. A disulphide bond is

not directly identified for ttADPGK from the sequence alignment. Other sequence motifs, however, are conserved in this unicellular protozoon, including the catalytic aspartate/arginine pair and the NEXE motif for recognition of Mg2+

. ADPGKs of insects (si, bm, dm) and sea urchin (sp) show multiple cysteine residues in the region in question (see Figure 80). The disulphide bond found in the mouse ADPGK still appears to be conserved, with the other cysteine residues being an additional pair, potentially forming another disulphide. The equivalent residues in the structure of mouse ADPGK, identified based on the sequence alignment in Figure 80 are structurally in close proximity to each other, making an additional disulphide at least in theory possible.

158 Figure 80: Sequence alignment of C-terminal region of ADPGK.

The sequence alignment shows the C-terminal region of ADPGKs from various eukaryotes and

archaea. Species: hs = H. sapiens, mm = M. musculus, pt = Pan troglodytes, bt = Bos taurus, ec =

Equus caballus, oc = Oryctolagus cuniculus, gg = Gallus gallus, dr = D. rerio, xt = Xenopus tropicalis,

ce = Caenorhabditis elegans, sp = Stronglycentrotus purpuratus, si = Solenopsis invicta, bm = Bombyx

mori, dm = Drosophila melanogaster, tt = Tetrahymena thermophila, tl = T. litoralis, pf = P. furiosus,

ph = P. horikoshii. Cysteine residues, including the cysteine residues homologous to those involved in

the formation of the disulphide bond observed in M. musculus ADPGK are highlighted with a yellow

box. The disulphide bond is indicated as a grey link between the cysteine residues for mmADPGK only. The disulphide bond is highly conserved in eukarya, but is absent in archaea.

The finding of the conserved disulphide (or multiple) is a bit of a surprise, as it is thought that ADPGK in human cells is anchored in the membrane of the endoplasmic reticulum facing the cytosol with its active site region, near where the disulphide bond is found (Kamiński et al. 2012). A protein with two cysteine residues in such close proximity that they are able to form a disulphide bond would be expected to be located in a compartment with oxidising environment, like the extracellular space or the lumen of the endoplasmic reticulum. However, intracellular disulphides do exist (Mallick et al. 2002), although they are usually of a transient nature and part of a

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- Q F I N P EA - - V - - - L D V L K L V - - - S L F H K Y K F K I N R V H Y H T I N L Q - M I C SN V N EWE S S R L A L V K S I T V A L Q E S EN K E R S F F D F E PQ K L K I S V P EQ - - I I L - MN V L - - - MD E T G I E R I H F H T Y G Y Y L A L T Q Y R G E E V RD - - A L L F A S L A A A A K AM K GN L E R I E Q I RD A L S V P T N E RA I V L E - M L K L - - - A K K T GV K R I H F H T Y G Y Y L A L T E Y K G EH V RD - - A L L F A A L A A A A K AM K GN I T S L E E I R EA T S V P V N E K A T Q V E - L L K L - - - I K E T GV K R I H F H T Y G Y Y L A L T R E K G EH V RD - - A L L F SA L A A A T K AM K GN I E K L S D I R EG L A V P I G EQG L E V E L RA PQ E FM T S H S EA - G S R I V L N PN K P V V EW H R E - - - G I S F H F T P V L V C K D P I R T V G L GD A I SA EG L F Y S E V H P H Y - - - - - L RA PQ E F T T S H L E S - G S R I V L N PD K P V V EW H R E - - - G I T F H F T P V L V C K D P V R T V G L GD A I SA EG L F Y S EA R P D - - - - L RA PQ E FM T S H S EA - G S R I V L N PN K P V V EW H R E - - - G I S F H F T P V L V C K D P I R T V G L GD A I SA EG L F Y S E V H P H Y - - - - - L K A PH E FM T S R L EA - G S R V V L N PN E P V V EW H R E - - - GV S F H F T P V L V C K D P V R T V G L GD A I SA EG L F Y S E V H P H L - - - - - L RA P R E FM T S H S EA - G S R I V V N PN K P V V EW H R E - - - G I S F H F T P V L V C K D P I R T V G L GD A I SA EG L F Y S E V H P H Y - - - - - L RA PQ E F V T S R L E V - G S R I V L N P R E P V V AW H R E - - - GV S F H F T P V L V C K D P V R T V G L GD A I SA EG L F Y S E V H P R - - - - L K A P L E F V T S Q I D A - P S K I S L N PD E P V V HW H R E - - - G I S F H F T P V L V C K D P V R T V G L GD A I SA EG L L Y S E I Y L Q - - - - L K A P L N F H S S F S E P - R E S L K V E P S R P V T VW R RG - - - N V S F H L T P V L V C K Q P L R T V G L GD A I SA EG L V F S E L T S E V - - - - - L K T P L E F S T S Q EQA - G S R V R V SA Q E P V A VW S R E - - - GV T F H F T P V L I C K D P V R T V G L GD A I SA EG L L F S EA T S QH P Y F - - I R T PA N F V L D K K - I - E K N YQ F EA H N P I A SW M R E - - - D V L F V F T P V L V C R L P S K T V G I D D A I SA T G L L Y SQ F Y R L N R P T HW L I MD D S F S V S M ED G - S R K I P L D V D K P V SCW K ED - - - D Y E I C V A P V L V C T Q V H K T A GGGD N I S S A G L S V Q I - - - - L I MD D S F S T S I V D - - G T R I A L N I D K P V SCW D E I L K V E - - S ED I S I Q V C V A P V L V C T QA SQ T A GGGD N I S S A G L V L Q I - - - - L L L D D S F S T T T D N D N T S R V F F E P T K P V A CW E E I L N GD - - - S V E I C I A P V L I C T EA Q L T A GA GD N I SA A G L V L Q V E K - - - - Q V L D D S F A T S A QA D - A P RM R I GA A S P V PCW R E Y I Q Y G RH R Q R L E V E I C V A P V L V C R EA R K T A GA GD N I SA SG L A A Q L - - - - P F F D D T F N I I Q SDQGQ K S I E F N P ED P V SCW N PH - - - K S I EC C V A PN I Q V I H PQ K T CG L GD N I S S T G L V Y H K V I S K Q - - - - - E E L E K E F T E F EN - - - G - - - L I DM V D RQ L A F V P T K I V A S P K S T V G I GD T I S S SA F V S E F GM R K R - - - - - E K L RA E Y G - I K E - - - G - - - I G E V EG YQ I A F I P T K I V A K P K S T V G I GD T I S S SA F I G E F S F T L - - - - K I L E K E F S - L RD - - - G - - - I G S I ED YQ L T F I P T K V V K K P K S T V G I GD T I S S SA F V S E F S L H - - - -

catalytic mechanism, e.g. in the case of thioredoxin (Prinz et al. 1997), or function as a redox sensor, e.g. in the case of the transcription factor OxyR (Choi et al. 2001). The membrane association of eukaryotic ADPGK is further supported by the predicted N-terminal signal peptide and reports of a cholesterol binding motif in the human ADPGK (Hulce et al. 2013). Based on the structure of the M. musculus

ADPGK, the deduction could be made that a potential transmembrane domain would be likely located in the part of the protein that was not crystallised – the first 50 amino acids - as the region proposed by Kamiński et al. (2012) would require a major rearrangement of the protein. The more commonly found cholesterol binding sites are the cholesterol recognition/interaction amino acid consensus sequence, short CRAC, and a similar, inverted motif called CARC (Li and Papadopoulos 1998; Baier et al. 2011). The consensus sequence for CRAC is (L/V)-X1−5-(Y)-X1−5-(K/R) and the

consensus sequence for CARC is (K/R)-X1−5-(Y/F)-X1−5-(L/V). The CARC motif

starts with a lysine or arginine residue with a positively charge side chain, which can form interactions with the hydroxyl group of cholesterol. The conserved aromatic phenylalanine or tyrosine residue in the centre of the motif can form a π-stack with cholesterol. The motif concludes with a branched, aliphatic amino acid, which can form van der Waals interactions with cholesterol. Other motifs are known, but they are less clearly defined. Also larger sequence separation between the defining residues has been observed and the aromatic residues can sometimes be substituted by tryptophan residues (Fantini and Barrantes 2013). The N-terminal region under discussion here is shown in Figure 81. The most compact possible CARC domain of human ADPGK would be 5-RGSAYAGFL-13. The tyrosine residue is not conserved