Influence of +1245 A/G MT1A Polymorphism on Advanced Glycation End-Products (Ages) In

SUPPLEMENTARY MATERIAL

Influence of +1245 A/G MT1A polymorphism on Advanced glycation end-products (AGEs) in elderly: effect of zinc supplementation

Giacconi R1*, Simm A2, Navarrete Santos A2, Costarelli L1, Malavolta M1, Mecocci P3, Piacenza F1, Basso A1, Fulop T4, Rink L5, Dedoussis G6, Kanoni S7, Herbein G8, Jajte J9, Mocchegiani E1. 1Centre of Nutrition and Ageing, Italian National Research Centre on Aging (INRCA), INRCA- IRCCS, 60121 Ancona, Italy 2 Martin-Luther University Halle-Wittenberg, Department of Cardiothoracic Surgery, Ernst-Grube Str. 40, D-06120 Halle/Saale, Germany 3 Section of Gerontology and Geriatrics, Department of Medicine, University of Perugia, Perugia, Italy 4Research Center on Aging, Department of Medicine, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Canada 5 Institute of Immunology, Medical Faculty, RWTH Aachen University, Aachen, Germany 6 Department of Dietetics and Nutritional Science, Harokopio University of Athens, Athens, Greece 7 Genetics of Complex Traits in Humans, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom 8 Pathogens and Inflammation Department, UPRES EA4266, CHRU Besancon, University of Franche-Comté, Besancon, France 9 Department of Toxicology, Faculty of Pharmacy, Medical University, Lodz, Poland

*Address correspondence to this author at the Trans. Res. Ctr. of Nutrition and Ageing, IRCCS- INRCA, Via Birarelli 8, 60121, Ancona, Italy; Tel: +39-071-8004213; Fax +39-071-206791; E- mail [email protected]

1 Table 5 Sequence alignment between human MT1A (NP_005937 metallothionein-1A) and MT proteins from some species of Vertebrata Subphylum metallothionein-1 [Danio rerio] Class: Actinopterygii; Order: Cypriniformes ref|NP_571150.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 38/60(63%) 1/60(1%) MDP C CA G+C C +CKC C+CT+CKKSCCSCCP C+KCA GC+CKG S SCC Sbjct 1 MDP-CECAKTGACNCGATCKCTNCQCTTCKKSCCSCCPSGCSKCASGCVCKGNSCGTSCC 59

PREDICTED: metallothionein-1C-like [Callorhinchus milii] Class: Chondrichthyes; Order: Chimaeriformes ref|XP_007905780.1|

Query 2 DPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 D C+C+ GG+C C G+C CK CKCTSCKKSCCSCCP C+KCAQGC+CKGASEKCSCC Sbjct 3 DAPCTCSEGGTCNCKGNCACKSCKCTSCKKSCCSCCPADCSKCAQGCVCKGASEKCSCC 61 44/59(75%) 0/59(0%) metallothionein A [Salmo salar] Class: Osteichthyes: Orders: Salmoniformes ref|NP_001117149.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKK-SCCSCCPMSCAKCAQGCICKGASEKCSC 59 MDP C C+ GSC C GSCKC C CTSCKK SCC CCP C+KCA GC+CKG + SC Sbjct 1 MDP-CECSKTGSCNCGGSCKCSNCACTSCKKASCCDCCPSGCSKCASGCVCKGKTCDTSC 59

Query 60 C 60 39/61(64%) 2/61(3%) C Sbjct 60 C 60

PREDICTED: metallothionein [Chrysemys picta bellii] Class : Reptilia; Orders: Testudines ref|XP_008165117.1|

Query 1 MDP-NCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICK-GASEKCS 58 2/62(3%) MDP NC CATGGSCTC GSCKCK C CTSC+KSCCSCCP C CA+GC+CK AS KCS 47/62(76%) Sbjct 1 MDPQNCPCATGGSCTCAGSCKCKNCSCTSCQKSCCSCCPPGCNNCAKGCVCKEPASNKCS 60

Query 59 CC 60 CC Sbjct 61 CC 62

2 PREDICTED: LOW QUALITY PROTEIN: metallothionein-1-like [Anolis carolinensis] Class: Sauropsida; Order: Squamata ref|XP_008106734.1|

Query 7 CATGGSCTCTGSCKCKECKCTSCKKSCCSC-CPMSCAKCAQGCICKGAS-EKCSCC 60 C +GG CTC+ + +CK CKC S KSCCS C M C+KCAQGCICKG KCS C Sbjct 15 CVSGGVCTCSSNFQCKHCKCKSYIKSCCSWPCLMGCSKCAQGCICKGPPLAKCSFC 70 39/56(69%) 2/56(3%) metallothionein [Xenopus laevis] Class: Amphibia; Order: Anura ref|NP_001081042.1| Query 1 MDP-NCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSC 59 MDP +C C TG SC+C +C C CKCTSCKKSCCSCCP C+KC+QGC C+ S+KCSC Sbjct 1 MDPQDCKCETGASCSCGTTCSCSNCKCTSCKKSCCSCCPAECSKCSQGCHCEKGSKKCSC 60

Query 60 C 60 40/61(66%) 1/61(1%) C Sbjct 61 C 61

PREDICTED: metallothionein-1-like [Columba livia] Class: Aves; Orders: Columbiformes ref|XP_005504830.1|

Query 4 NCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKG-ASEKCSCC 60 +C CA GG+CTC +CKCK CKCTSCKK CCSCCP CAKCAQGC+CKG S KCSCC 43/58(74%) 1/58(1%) Sbjct 5 DCPCAAGGTCTCGDNCKCKNCKCTSCKKGCCSCCPAGCAKCAQGCVCKGPPSAKCSCC 62 metallothionein [Gallus gallus] Class: Aves; Orders: Galliformes ref|NP_990606.1|

Query 1 MDP-NCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICK-GASEKCS 58 MDP +C+CA G SC+C GSCKCK C+C SC+KSCCSCCP C CA+GC+CK AS KCS Sbjct 1 MDPQDCTCAAGDSCSCAGSCKCKNCRCRSCRKSCCSCCPAGCNNCAKGCVCKEPASSKCS 60

Query 59 CC 60 42/62(68%) 2/62(3%) CC Sbjct 61 CC 62

3 PREDICTED: metallothionein-1A-like [Macaca fascicularis] Class: Mammals; Orders: Primates ref|XP_005592030.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSC 59 MDPNCSCATGGSCTC+GSCKCKECKCTSCKKS CSCCP+SCA+CAQGC+CKGASEKCSC Sbjct 1 MDPNCSCATGGSCTCSGSCKCKECKCTSCKKSRCSCCPVSCARCAQGCVCKGASEKCSC 59 54/59(92%) 0/59(0%)

PREDICTED: metallothionein-1-like [Papio anubis] Class: Mammals; Orders: Primates ref|XP_003916961.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC+TGGSCTC GSCKCKE TSCKKSCCSCCP+ CAKCAQGC+CKG S++CSCC Sbjct 1 MDPNCSCSTGGSCTCAGSCKCKERTFTSCKKSCCSCCPVGCAKCAQGCVCKGTSDRCSCC 60

Query 61 A 61 50/61(82%) 0/61(0%) A Sbjct 61 A 61

PREDICTED: metallothionein-1-like [Leptonychotes weddellii] Class: Mammals; Orders: Carnivora ref|XP_006749274.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC+TGGSCTC GSCKCKECKCTSCKKSCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDPNCSCSTGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60

Query 61 A 61 56/61(92%) 0/61(0%) A Sbjct 61 A 61 metallothionein-1 [Canis lupus familiaris] Class: Mammals; Orders: Carnivora ref|NP_001003173.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 55/61(90%) 0/61(0%) MDP+CSC+TGGSCTC GSCKCKECKCTSCKKSCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDPDCSCSTGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60

Query 61 A 61 4 A Sbjct 61 A 61

PREDICTED: metallothionein-2 [Trichechus manatus latirostris] Class: Mammals; Orders: Sirenia ref|XP_004371623.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSCATGGSC C GSCKCKECKCTSCK+SCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDPNCSCATGGSCACAGSCKCKECKCTSCKRSCCSCCPVGCAKCAQGCICKGASDKCSCC 60

Query 61 A 61 55/61(90%) 0/61(0%) A Sbjct 61 A 61

PREDICTED: metallothionein-1-like [Panthera tigris altaica] Class: Mammals; Orders: Carnivora ref|XP_007090251.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDP+CSCATGGSCTC GSC CKEC+CTSCKKSCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDPDCSCATGGSCTCAGSCTCKECRCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60

Query 61 A 61 54/61(89%) 0/61(0%) A Sbjct 61 A 61

PREDICTED: metallothionein-2-like [Loxodonta africana] Class: Mammals; Orders: Proboscidea ref|XP_003416350.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MD NCSCATGGSC C GSCKCKECKCTSC+KSCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDSNCSCATGGSCACPGSCKCKECKCTSCRKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60

Query 61 A 61 54/61(89%) 0/61(0%) A Sbjct 61 A 61

5 PREDICTED: metallothionein-1A-like [Equus caballus] Class: Mammals; Orders: Perissodactyla ref|XP_003364650.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC TGGSCTC GSCKCKEC+CTSCKKSCC CCP+ CA+CAQGCICKGAS+KCSCC Sbjct 1 MDPNCSCPTGGSCTCAGSCKCKECRCTSCKKSCCPCCPVGCARCAQGCICKGASDKCSCC 60 53/61(87%) 0/61(0%) Query 61 A 61 A Sbjct 61 A 61 PREDICTED: metallothionein-2 [Vicugna pacos] Class: Mammals; Orders: Artiodactyla ref|XP_006214597.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC++G SC+C GSCKCKECKCTSCKKSCCSCCP+ CAKCAQGCICKGAS+KCSCC Sbjct 1 MDPNCSCSSGDSCSCPGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCC 60 53/61(87%) 0/61(0%) Query 61 A 61 A Sbjct 61 A 61 PREDICTED: metallothionein-1A-like [Pteropus alecto] Class: Mammals; Orders: Chiroptera ref|XP_006908832.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC TGGSC+CTGSCKCK+C+CTSCKKSCCSCCP+ CA+CAQGC+CKGAS+KCSCC Sbjct 1 MDPNCSCPTGGSCSCTGSCKCKDCRCTSCKKSCCSCCPVGCARCAQGCVCKGASDKCSCC 60 52/61(85%) 0/61(0%) Query 61 A 61 A Sbjct 61 A 61 PREDICTED: metallothionein-1E-like [Physeter catodon] Class: Mammals; Orders: Cetacea ref|XP_007106457.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 51/61(84%) 0/61(0%) MDPNCSC+TGGSC+C GSC CK C+CTSCKKSCCSCCP+ CAKCAQGC+CKGAS+KCSCC Sbjct 39 MDPNCSCSTGGSCSCAGSCTCKACRCTSCKKSCCSCCPVGCAKCAQGCVCKGASDKCSCC 98

Query 61 A 61 A

6 Sbjct 99 A 99 metallothionein-1A [Sus scrofa] Class: Mammals; Orders: Artiodactyla ref|NP_001001266.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC TGGSC+C GSC CK C+CTSCKKSCCSCCP CA+CAQGCICKGAS+KCSCC Sbjct 1 MDPNCSCPTGGSCSCAGSCTCKACRCTSCKKSCCSCCPAGCARCAQGCICKGASDKCSCC 60

Query 61 A 61 A 51/61(84%) 0/61(0%) Sbjct 61 A 61

PREDICTED: metallothionein-1A [Ovis aries] Class: Mammals; Orders: Artiodactyla ref|XP_004015045.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC TGGSC+C GSC CK C+C SCKKSCCSCCP+ CAKCAQGC+CKGAS+KCSCC Sbjct 1 MDPNCSCPTGGSCSCAGSCTCKACRCPSCKKSCCSCCPVGCAKCAQGCVCKGASDKCSCC 60 50/61(82%) 0/61(0%) Query 61 A 61 A Sbjct 61 A 61 metallothionein-1 [Rattus norvegicus] Class: Mammals; Orders: Rodentia ref|NP_620181.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC+TGGSCTC+ SC CK CKCTSCKKSCCSCCP+ C+KCAQGC+CKGAS+KC+CC Sbjct 1 MDPNCSCSTGGSCTCSSSCGCKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGASDKCTCC 60

Query 61 A 61 50/61(82%) 0/61(0%) A Sbjct 61 A 61

7 metallothionein-1 [Mus musculus] Class: Mammals; Orders: Rodentia ref|NP_038630.1|

Query 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCC 60 MDPNCSC+TGGSCTCT SC CK CKCTSCKKSCCSCCP+ C+KCAQGC+CKGA++KC+CC Sbjct 1 MDPNCSCSTGGSCTCTSSCACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGAADKCTCC 60

Query 61 A 61 50/61(82%) 0/61(0%) A Sbjct 61 A 61

Query= NP_005937 metallothionein-1A sequence Sbjct= Ref sequence

Table 5 reports NP_005937 metallothionein-1A [Homo sapiens] alignments with MT isoforms from representative species of various vertebrate classes and orders Sequence alignments were performed using protein BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?PAGE=Proteins)

8 I-TASSER results 1: Submitted Sequence MT1A Lys51 (K) variant

>protein 1 MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCCA61

Predicted Secondary Structure

20 40 60 | | | Sequence MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCCA Prediction CCCCCCCCCCCCSSCCCCSSCCCCCCCCCCCCCCCCCCCCHHHHCCCCSSCCCCCCCCCCC Conf.Score 9887624799867067864407862775365535667825667735742427776654349

Predicted Solvent Accessibility

20 40 60 | | | Sequence MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCCA Prediction 8647151454341413441416716144146310421344034104311143446524227

Values range from 0 (buried residue) to 9 (highly exposed residue)

Predicted normalized B-factor

9 Top 10 templates used by I-TASSER

Rank PDB Iden1 Iden2 Cov. Norm. Download 20 40 60

Hit Z-score Align. | | | Sec.Str CCCCCCCCCCCCSSCCCCSSCCCCCCCCCCCCCCCCCCCCHHHHCCCCSSCCCCCCCCCCC Seq MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICKGASEKCSCCA 1 4mt2A 0.85 0.85 1.00 5.48 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 2 4mt2A 0.85 0.85 1.00 1.26 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 3 4mt2A 0.85 0.85 1.00 6.17 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 4 4mt2A 0.85 0.85 1.00 7.40 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 5 4mt2A 0.85 0.85 1.00 5.39 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 6 4mt2A 0.85 0.85 1.00 7.02 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 7 4mt2A 0.85 0.85 0.98 6.72 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCC- 8 4mt2A 0.85 0.00 1.00 2.45 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 9 4mt2A 0.85 0.85 1.00 4.29 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 10 4mt2A 0.85 0.85 1.00 1.57 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA (a) All the residues are colored in black; however, those residues in template which are identical to the residue in the query sequence are highlighted in color. Coloring scheme is based on the property of amino acids, where polar are brightly coloured while non-polar residues are colored in dark shade. (more about the colors used) (b) Rank of templates represents the top ten threading templates used by I-TASSER. (c) Ident1 is the percentage sequence identity of the templates in the threading aligned region with the query sequence. (d) Ident2 is the percentage sequence identity of the whole template chains with query sequence. (e) Cov. represents the coverage of the threading alignment and is equal to the number of aligned residues divided by the length of query protein. (f) Norm. Z-score is the normalized Z-score of the threading alignments. Alignment with a Normalized Z-score >1 mean a good alignment and vice versa. (g) Download Align. provides the 3D structure of the aligned regions of the threading templates. (h) The top 10 alignments reported above (in order of their ranking) are from the following threading programs:

10 1: MUSTER 2: FFAS-3D 3: SPARKS-X 4: HHSEARCH2 5: HHSEARCH I 6: Neff-PPAS 7: HHSEARCH 8: pGenTHREADER 9: wdPPAS 10: PROSPECT2

Top 5 Models predicted by I-TASSER

11 Generated Estimated local accuracy of models 3D models

Model 1  C- score=1.60 (Read more about C- score)  Estimated TM-score =0.94±0.05  Estimated RMSD = 0.5±0.5Å

Model 2

C-score = -5

Model 3

C-score = -1.32

Model 4

C-score = -5

Generated Estimated local accuracy of models 3D models

Model 1  C- score=1.60 (Read more about C- score)  Estimated TM-score =0.94±0.05  Estimated RMSD = 0.5±0.5Å

Model 2

C-score = -5

Model 3

C-score = -1.32(a) Query structure is shown in cartoon, while the structural analog is displayed using backbone trace. (b) Ranking of proteins is based on TM-score of the structural alignment between the query structure and known structures in the PDB library. (c) RMSDa is the RMSD between residues that are structurally aligned by TM-align. (d) IDENa is the percentage sequence identity in the structurally aligned region. (e) Cov. represents the coverage of the alignment by TM-align and is equal to the number of structurally aligned residues divided by length of the query protein.

Model 1 shows the better estimated local accuracy (see RSQ-1 values in Table 7) RSQ=Residue-Specific Quality of models defined as the estimated deviation of the residue on the model from the native structure of the protein) For more information about the local accuracy and normalized B-factor predictions, please refer to the following article:Jianyi Yang and Yang zhang, ResQ: A unified algorithm for estimating B-factor and residue-specific quality of protein structure prediction, submitted, (2014).

Model 4

C-score I-TASSER = -5 results 2: Submitted Sequence MT1A Arg51 (R) variant

>protein 1MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICRGASEKCSCCA61

Predicted Secondary Structure

20 40 60 | | | Sequence MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICRGASEKCSCCA Prediction CCCCCCCCCCCCSSCCCCSSSCCCCCCCCCCCCCCCCCCCHHHHCCCCSSCCCCCCCCCCC Conf.Score 9888625799857068864526763774365535767714667725642437776654349

Predicted Solvent Accessibility

20 40 60 | | | Sequence MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICRGASEKCSCCA Prediction 8647161444341413640415716244146320421344034104311143446524227

13 Values range from 0 (buried residue) to 9 (highly exposed residue)

Predicted normalized B-factor

Top 10 templates used by I-TASSER

Rank PDB Iden1 Iden2 Cov. Norm. Download 20 40 60

Hit Z-score Align. | | | Sec.Str CCCCCCCCCCCCSSCCCCSSSCCCCCCCCCCCCCCCCCCCHHHHCCCCSSCCCCCCCCCCC Seq MDPNCSCATGGSCTCTGSCKCKECKCTSCKKSCCSCCPMSCAKCAQGCICRGASEKCSCCA 1 4mt2A 0.84 0.84 1.00 5.40 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 2 4mt2A 0.84 0.84 1.00 1.25 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA

14 3 4mt2A 0.84 0.84 1.00 6.23 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 4 4mt2A 0.84 0.84 1.00 7.40 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 5 4mt2A 0.84 0.84 1.00 5.41 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 6 4mt2A 0.84 0.84 1.00 7.05 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 7 4mt2A 0.83 0.84 0.98 6.72 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCC- 8 4mt2A 0.84 0.00 1.00 2.45 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 9 4mt2A 0.84 0.84 1.00 4.21 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA 10 4mt2A 0.84 0.84 1.00 1.55 Download MDPNCSCATDGSCSCAGSCKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA (a) All the residues are colored in black; however, those residues in template which are identical to the residue in the query sequence are highlighted in color. Coloring scheme is based on the property of amino acids, where polar are brightly coloured while non-polar residues are colored in dark shade. (more about the colors used) (b) Rank of templates represents the top ten threading templates used by I-TASSER. (c) Ident1 is the percentage sequence identity of the templates in the threading aligned region with the query sequence. (d) Ident2 is the percentage sequence identity of the whole template chains with query sequence. (e) Cov. represents the coverage of the threading alignment and is equal to the number of aligned residues divided by the length of query protein. (f) Norm. Z-score is the normalized Z-score of the threading alignments. Alignment with a Normalized Z-score >1 mean a good alignment and vice versa. (g) Download Align. provides the 3D structure of the aligned regions of the threading templates. (h) The top 10 alignments reported above (in order of their ranking) are from the following threading programs: 1: MUSTER 2: FFAS-3D 3: SPARKS-X 4: HHSEARCH2 5: HHSEARCH I 6: Neff-PPAS 7: HHSEARCH 8: pGenTHREADER 9: wdPPAS 10: PROSPECT2

15 Top 5 Models predicted by I-TASSER

16 Generated Estimated local accuracy of models 3D models

Model 1  C- score=1.53 (Read more about C- score)  Estimated TM-score = 0.93±0.06  Estimated RMSD = 0.5±0.5Å

Model 2  C-score = -5

17 Model 3  C-score = (a) Query structure is shown in cartoon, while the structural analog is displayed using backbone trace. (b) Ranking of proteins is based on TM-score of the structural alignment between the query structure and known structures in the PDB library. (c) RMSDa is the RMSD between residues that are structurally aligned by TM-align. (d) IDENa is the percentage sequence identity in the structurally aligned region. (e) Cov. represents the coverage of the alignment by TM-align and is equal to the number of structurally aligned residues divided by length of the query protein.

Model 1 shows the better estimated local accuracy (see RSQ-1 values in Table 8) RSQ=Residue-Specific Quality of models defined as the estimated deviation of the residue on the model from the native structure of the protein) For more information about the local accuracy and normalized B-factor predictions, please refer to the following article:Jianyi Yang and Yang zhang, ResQ: A unified algorithm for estimating B-factor and residue-specific quality of protein structure prediction, submitted, (2014).

18 Table 7 B-factor and local structure quality estimation in I-TASSER structure modeling of MT1A Lys51 isoform

#RES SS SA COV BFP RSQ_1 RSQ_2 RSQ_3 RSQ_4 1 C E 0.46 2.13 0.78 8.98 7.42 9.50 2 C E 0.53 1.49 0.92 7.70 6.92 8.71 3 C E 0.56 0.82 0.68 7.19 7.07 9.11 4 C E 0.58 0.30 0.60 9.11 6.52 8.48 5 C B 0.64 -0.27 0.43 6.62 5.53 6.46 6 C E 0.64 -0.23 0.42 6.20 5.75 7.09 7 C B 0.66 -0.35 0.39 6.01 5.30 6.12 8 C E 0.67 -0.23 0.46 6.81 5.70 5.85 9 C E 0.64 -0.14 0.56 6.03 5.82 5.60 10 C E 0.69 -0.12 0.62 6.16 5.87 6.04 11 C E 0.71 -0.13 0.61 6.34 5.73 5.79 12 C E 0.69 -0.10 0.54 5.39 5.40 5.12 13 S B 0.68 -0.40 0.34 5.14 4.92 5.25 14 S E 0.69 -0.36 0.41 5.22 5.32 5.33 15 C B 0.69 -0.40 0.40 5.25 5.19 5.27 16 C E 0.69 -0.18 0.46 5.26 5.40 5.30 17 C E 0.69 0.01 0.53 5.47 5.43 5.34 18 C E 0.67 -0.15 0.55 5.38 5.47 5.33 19 S B 0.66 -0.48 0.45 5.16 5.06 5.19 20 S E 0.66 -0.37 0.50 5.23 5.35 5.28 21 C B 0.64 -0.42 0.37 5.18 5.07 5.30 22 C E 0.69 -0.06 0.46 5.23 5.31 5.40 23 C E 0.69 -0.10 0.42 5.21 5.25 5.36 24 C B 0.67 -0.36 0.34 5.14 5.06 5.30 25 C E 0.68 -0.26 0.41 5.10 5.40 5.26 26 C B 0.69 -0.28 0.40 5.03 5.09 5.29 27 C E 0.69 -0.13 0.45 4.99 5.41 5.25 28 C E 0.67 -0.54 0.45 5.04 5.42 5.22 29 C B 0.64 -0.41 0.39 5.03 5.22 5.06 30 C E 0.64 -0.20 0.56 5.59 5.84 5.52 31 C E 0.64 -0.09 0.47 5.45 5.20 5.25 32 C E 0.69 -0.29 0.48 5.11 5.27 5.19 33 C B 0.68 -0.53 0.52 5.07 5.27 5.23 34 C B 0.64 -0.65 0.45 5.19 5.21 5.44 35 C E 0.66 -0.38 0.49 5.33 5.62 5.48 36 C B 0.66 -0.45 0.42 5.42 5.17 5.66 37 C B 0.66 -0.46 0.39 5.46 5.19 5.62 38 C E 0.67 -0.40 0.46 5.52 5.58 5.50 39 C E 0.69 -0.16 0.53 5.28 5.57 5.32 40 C E 0.69 -0.11 0.52 5.49 5.69 5.57 41 H B 0.69 -0.28 0.45 5.54 5.40 5.77 42 H E 0.67 -0.19 0.48 5.63 5.49 6.00 43 H E 0.66 -0.01 0.47 5.55 5.40 6.08 44 H B 0.67 -0.26 0.39 5.48 5.05 5.76 45 C B 0.66 -0.43 0.49 5.57 5.32 5.73 46 C E 0.66 -0.17 0.61 5.74 5.91 6.03

19 47 C E 0.69 -0.27 0.52 6.08 5.56 5.90 48 C B 0.69 -0.68 0.42 5.39 5.24 5.68 49 S B 0.66 -0.42 0.39 5.28 5.17 5.60 50 S B 0.64 -0.22 0.37 5.78 5.02 6.09 51 C E 0.61 0.33 0.50 5.67 5.53 5.99 52 C E 0.68 0.58 0.58 6.77 5.90 6.11 53 C E 0.66 0.67 0.59 6.40 5.68 5.95 54 C E 0.67 0.58 0.59 6.09 5.65 6.29 55 C E 0.69 0.65 0.61 7.95 5.72 6.38 56 C E 0.69 0.27 0.53 7.07 5.51 5.78 57 C B 0.70 -0.05 0.45 6.77 5.50 6.71 58 C E 0.68 -0.05 0.57 8.99 6.04 8.61 59 C E 0.67 0.08 0.55 9.25 6.16 10.13 60 C E 0.64 0.70 0.51 9.87 5.81 9.38 61 C E 0.57 1.75 0.82 12.78 6.81 11.65

The black bold type indicates the Cys residues The blue bold type indicates the Arg residue The letter in yellow shows the change of Predicted solvent accessibility or Predicted secondary structure as compared to MT1A Arg51 isoform

RES: Residue number of aminoacid SS: Predicted secondary structure: C - random coil; H - alpha-helix; S - beta-strand SA: Predicted solvent accessibility at 25% cutoff: E - exposed; B - buried COV: Threading alignment coverage defined as the number of threading alignments on the residue divided by the number of total threading programs BFP: Predicted normalized B-factor RSQ_*: Residue-Specific Quality of models defined as the estimated deviation of the residue on the model from the native structure of the protein

20 Table 8 B-factor and local structure quality estimation in I-TASSER structure modeling of MT1A Arg51 isoform

#RES SS SA COV BFP RSQ_1 RSQ_2 RSQ_3 1 C E 0.46 2.15 0.76 9.87 9.52 2 C E 0.55 1.40 0.93 8.20 8.40 3 C E 0.59 0.82 0.75 7.36 9.40 4 C E 0.58 0.33 0.67 7.21 10.09 5 C B 0.64 -0.22 0.48 6.77 7.90 6 C E 0.65 -0.15 0.43 7.17 7.32 7 C B 0.65 -0.28 0.41 5.45 5.88 8 C E 0.66 -0.22 0.54 6.53 6.79 9 C E 0.64 -0.16 0.62 6.38 6.16 10 C E 0.69 -0.14 0.65 5.85 6.21 11 C E 0.69 -0.18 0.64 7.85 6.78 12 C E 0.69 -0.13 0.55 5.48 5.87 13 S B 0.68 -0.39 0.33 4.90 5.46 14 S E 0.68 -0.30 0.43 5.25 5.45 15 C B 0.68 -0.34 0.40 5.20 5.41 16 C E 0.68 -0.15 0.47 5.32 5.43 17 C E 0.68 0.05 0.54 5.47 5.40 18 C E 0.68 -0.15 0.53 5.37 5.51 19 S B 0.68 -0.52 0.43 5.12 5.27 20 S E 0.68 -0.40 0.52 5.14 5.32 21 S B 0.67 -0.46 0.37 5.09 5.24 22 C E 0.70 -0.12 0.47 5.14 5.33 23 C E 0.71 -0.07 0.42 5.06 5.18 24 C B 0.67 -0.32 0.39 5.07 5.14 25 C E 0.69 -0.16 0.42 4.95 5.08 26 C E 0.69 -0.18 0.40 5.01 5.16 27 C E 0.71 -0.06 0.45 5.01 5.13 28 C E 0.68 -0.40 0.44 5.07 5.19 29 C B 0.66 -0.24 0.39 5.02 5.07 30 C E 0.65 -0.08 0.54 5.45 5.38 31 C E 0.67 -0.00 0.46 5.38 5.23 32 C E 0.70 -0.27 0.46 5.21 4.96 33 C B 0.69 -0.51 0.51 5.20 4.90 34 C B 0.67 -0.61 0.42 5.09 5.16 35 C E 0.66 -0.35 0.47 5.28 5.39 36 C B 0.63 -0.39 0.44 5.34 5.43 37 C B 0.64 -0.49 0.43 5.27 5.31 38 C E 0.66 -0.37 0.44 5.28 5.29 39 C E 0.68 -0.19 0.51 5.13 5.11 40 C E 0.69 -0.14 0.52 5.24 5.26 41 H B 0.67 -0.27 0.43 5.28 5.33

21 42 H E 0.64 -0.12 0.47 5.39 5.48 43 H E 0.64 0.04 0.43 5.33 5.46 44 H B 0.66 -0.20 0.36 5.16 5.23 45 C B 0.64 -0.39 0.48 5.19 5.21 46 C E 0.66 -0.14 0.55 5.63 5.52 47 C E 0.69 -0.25 0.51 5.69 5.62 48 C B 0.68 -0.70 0.37 5.27 5.35 49 S B 0.67 -0.50 0.36 5.74 5.45 50 S B 0.66 -0.29 0.34 5.93 5.10 51 C E 0.63 0.29 0.51 6.61 5.70 52 C E 0.67 0.48 0.56 7.36 5.63 53 C E 0.65 0.56 0.56 5.77 5.61 54 C E 0.66 0.53 0.57 6.37 5.75 55 C E 0.68 0.67 0.61 6.81 5.99 56 C E 0.69 0.37 0.54 7.08 6.05 57 C E 0.69 0.09 0.45 7.17 5.88 58 C E 0.67 0.08 0.56 9.92 6.79 59 C E 0.66 0.18 0.52 9.55 6.70 60 C E 0.64 0.65 0.50 9.73 6.40 61 C E 0.56 1.64 0.81 12.54 6.80

The black bold type indicates the Cys residues The blue bold type indicates the Arg residue The letter in yellow shows the change of Predicted solvent accessibility or Predicted secondary structure as compared to MT1A Lys51 isoform

RES: Residue number of aminoacid SS: Predicted secondary structure: C - random coil; H - alpha-helix; S - beta-strand SA: Predicted solvent accessibility at 25% cutoff: E - exposed; B - buried COV: Threading alignment coverage defined as the number of threading alignments on the residue divided by the number of total threading programs BFP: Predicted normalized B-factor RSQ_*: Residue-Specific Quality of models defined as the estimated deviation of the residue on the model from the native structure of the protein

Bibliography

1) Yang Zhang. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9:40 (2008). 2) Ambrish Roy, Alper Kucukural, Yang Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, vol 5, 725-738 (2010). 3) Ambrish Roy, Jianyi Yang, Yang Zhang. COFACTOR: an accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Research, vol 40, W471-W477 (2012). 4) Jianyi Yang and Yang Zhang, ResQ: A unified algorithm for estimating B-factor and residue- specific quality of protein structure prediction, submitted, (2014).

22 Supplementary Figures

Fig1A tridimensional structure (model 1) of MT1A Lys51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 7

23 Fig1B tridimensional structure (model 2) of MT1A Lys51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 7

24 Fig1C tridimensional structure (model 3) of MT1A Lys51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 7

25 Fig1D tridimensional structure (model 4) of MT1A Lys51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 7

26 Fig2A tridimensional structure (model 1) of MT1A Arg51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 8

27 Fig2B tridimensional structure (model 2) of MT1A Arg51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 8

28 Fig2C tridimensional structure (model 3) of MT1A Arg51 isoform calculated by I-TASSER online Structure & Function Prediction (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) The Residue-Specific Quality (RSQ1) of the model, defined as the estimated deviation of the residue on the model from the native structure of the protein, is reported in Table 8