Pages 1–21 1tt5 Evolutionary trace report by report maker January 22, 2010

4 Notes on using trace results 19 4.1 Coverage 19 4.2 Known substitutions 19 4.3 Surface 19 4.4 Number of contacts 19 4.5 Annotation 19 4.6 Mutation suggestions 19

5 Appendix 19 5.1 File formats 19 5.2 Color schemes used 19 5.3 Credits 20 5.3.1 Alistat 20 5.3.2 CE 20 5.3.3 DSSP 20 5.3.4 HSSP 20 5.3.5 LaTex 20 5.3.6 Muscle 20 5.3.7 Pymol 20 5.4 Note about ET Viewer 20 5.5 Citing this work 20 CONTENTS 5.6 About report maker 20 5.7 Attachments 20 1 Introduction 1

2 Chain 1tt5A 1 1 INTRODUCTION 2.1 Q13564 overview 1 From the original Protein Data Bank entry (PDB id 1tt5): 2.2 Multiple sequence alignment for 1tt5A 1 Title: Structure of appbp1-uba3-ubc12n26: a unique e1-e2 interac- 2.3 Residue ranking in 1tt5A 2 tion required for optimal conjugation of the ubiquitin-like protein 2.4 Top ranking residues in 1tt5A and their position on nedd8 the structure 2 Compound: Mol id: 1; molecule: amyloid protein-binding pro- 2.4.1 Clustering of residues at 25% coverage. 2 tein 1; chain: a, c; synonym: appbp1; engineered: yes; mol id: 2; 2.4.2 Overlap with known functional surfaces at molecule: ubiquitin-activating enzyme e1c isoform 1; chain: b, d; 25% coverage. 3 fragment: residues 33-463; synonym: uba3, nedd8-activating enzyme 2.4.3 Possible novel functional surfaces at 25% huba3; engineered: yes; mol id: 3; molecule: ubiquitin-conjugating coverage. 6 enzyme e2 m; chain: e, f; fragment: residues 1-26; synonym: ubc12n26, ubiquitin-protein ligase m, ubiquitin carrier protein m, 3 Chain 1tt5B 9 nedd8-conjugating enzyme ubc12; engineered: yes 3.1 Q8TBC4 overview 9 Organism, scientific name: Homo Sapiens; 3.2 Multiple sequence alignment for 1tt5B 10 1tt5 contains unique chains 1tt5A (522 residues) and 1tt5B (414 3.3 Residue ranking in 1tt5B 10 residues) 1tt5C is a homologue of chain 1tt5A. 1tt5D is a homo- 3.4 Top ranking residues in 1tt5B and their position on logue of chain 1tt5B. Chains 1tt5E and 1tt5F are too short to permit the structure 10 statistically significant analysis, and were treated as a peptide ligands. 3.4.1 Clustering of residues at 25% coverage. 10 3.4.2 Overlap with known functional surfaces at 2 CHAIN 1TT5A 25% coverage. 11 3.4.3 Possible novel functional surfaces at 25% 2.1 Q13564 overview coverage. 16 From SwissProt, id Q13564, 97% identical to 1tt5A:

1 Lichtarge lab 2006 Description: Amyloid protein-binding protein 1 (Amyloid beta pre- cursor protein- binding protein 1, 59 kDa) (APP-BP1) (Protoonco- gene protein 1) (HPP1). Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini; Hominidae; Homo. Function: The dimeric enzyme activates NEDD8 by first adenylating its C-terminal glycine residue with ATP and thereafter linking this residue to the side chain of a cysteine residue on UBE1C, yielding a NEDD8-UBE1C thiolester and free AMP. Necessary for cell cycle progression through the S-M checkpoint. Overexpression of APPBP1 causes apoptosis through deregulation of NEDD8 conjugation. Enzyme regulation: Binding of TP53BP2 to the regulatory subunit APPBP1 decreases neddylation activity. Pathway: NEDD8 conjugation; first step. Subunit: Heterodimer of UBE1C and APPBP1. The complex binds Fig. 1. Residues 1-273 in 1tt5A colored by their relative importance. (See NEDD8. Binds APP and TP53BP2. Appendix, Fig.17, for the coloring scheme.) Subcellular location: Colocalizes with APP in lipid rafts. Tissue specificity: Ubiquitous in fetal tissues. Expressed throughout the adult brain. Miscellaneous: APPBP1 and UBE1C correspond to the N-terminal and the C-terminal part of yeast UBA3. In yeast the two subunits form a single polypeptide chain. Similarity: Belongs to the ubiquitin-activating E1 family. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed.

2.2 Multiple sequence alignment for 1tt5A For the chain 1tt5A, the alignment 1tt5A.msf (attached) with 81 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as Fig. 2. Residues 274-534 in 1tt5A colored by their relative importance. (See duplicate sequences were removed. It can be found in the attachment Appendix, Fig.17, for the coloring scheme.) to this report, under the name of 1tt5A.msf. Its statistics, from the alistat program are the following: 2.3 Residue ranking in 1tt5A Format: MSF The 1tt5A sequence is shown in Figs. 1–2, with each residue colored Number of sequences: 81 according to its estimated importance. The full listing of residues Total number of residues: 34273 in 1tt5A can be found in the file called 1tt5A.ranks sorted in the Smallest: 149 attachment. Largest: 522 Average length: 423.1 2.4 Top ranking residues in 1tt5A and their position on Alignment length: 522 the structure Average identity: 34% In the following we consider residues ranking among top 25% of resi- Most related pair: 99% dues in the protein . Figure 3 shows residues in 1tt5A colored by their Most unrelated pair: 6% importance: bright red and yellow indicate more conserved/important Most distant seq: 35% residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Furthermore, <1% of residues show as conserved in this ali- 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the gnment. top 25% of all residues, this time colored according to clusters they The alignment consists of 23% eukaryotic ( 7% vertebrata, 1% belong to. The clusters in Fig.4 are composed of the residues listed arthropoda, 9% fungi, 4% plantae) sequences. (Descriptions of in Table 1. some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1tt5A.descr.

2 Table 1. continued cluster size member color residues red 56 184,188,192,209,210,211,212 213,214,217,218,220,222,225 261,262,263,264,265,267,268 271,272,274,304,305,308,310 311,312,314,323,324,325,326 327,330,331,332,334,336,339 342,343,346,349,350,353,380 383,384,385,386,387,390,392 blue 51 9,12,13,14,15,16,17,18,19,20 21,24,25,28,31,42,44,46,47 48,49,51,52,53,55,57,93,95 96,97,100,155,157,158,160 489,491,493,498,499,501,502 503,504,506,507,510,512,513 515,522 yellow 5 69,71,73,74,76 green 3 37,61,86 purple 2 34,123 azure 2 234,240 Fig. 3. Residues in 1tt5A, colored by their relative importance. Clockwise: turquoise 2 518,519 front, back, top and bottom views. brown 2 481,484

Table 1. Clusters of top ranking residues in 1tt5A.

2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1tt5B.Table 2 lists the top 25% of residues at the interface with 1tt5B. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 4.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 157 G G(100) 0.00 3/3 4.35 504 E .(24) 0.01 12/0 3.21 E(75) 507 K .(24) 0.01 27/0 2.58 K(75) 331 D .(25) 0.02 34/1 2.58 D(74) 47 K K(96) 0.03 29/0 3.12 S(1) .(2) Fig. 4. Residues in 1tt5A, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for 484 R .(25) 0.03 108/17 2.68 the coloring scheme). Clockwise: front, back, top and bottom views. The R(71) corresponding Pymol script is attached. S(2) 44 E E(92) 0.04 14/0 3.20 R(1) Table 1. .(2) cluster size member Q(2) color residues continued in next column continued in next column

3 Table 2. continued Table 2. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) X(1) F(1) 332 M .(25) 0.04 15/15 3.14 .(2) M(71) 334 A .(25) 0.09 26/26 3.30 I(1) A(45) F(1) S(24) 499 G .(25) 0.04 24/24 3.28 T(1) G(72) C(2) S(1) 336 S .(25) 0.09 46/15 2.48 51 L L(95) 0.05 45/12 3.02 S(39) .(2) T(34) I(1) 343 Q .(25) 0.09 2/0 4.73 F(1) Q(66) 96 L L(95) 0.05 7/1 3.92 A(3) F(2) P(1) M(2) K(1) 481 E .(25) 0.06 39/4 2.80 R(1) E(71) 491 H .(25) 0.09 44/0 2.82 D(1) H(69) S(1) N(1) 13 Y Y(92) 0.07 9/4 3.93 P(2) .(6) Q(1) K(1) 19 L V(23) 0.11 37/1 3.50 15 R R(92) 0.07 98/28 2.86 L(55) .(6) I(14) N(1) .(4) 16 Q Q(92) 0.07 32/9 2.99 X(1) .(6) 95 E E(91) 0.11 33/14 2.96 E(1) Q(2) 18 R Y(23) 0.07 61/5 2.99 .(2) R(70) A(1) .(4) R(1) X(1) D(1) 20 W L(23) 0.07 11/0 3.80 339 Y .(25) 0.11 27/0 3.37 W(70) Y(65) .(4) F(7) X(1) R(1) 211 H .(24) 0.07 14/1 3.77 12 K L(23) 0.12 39/28 3.45 H(64) K(56) K(8) R(12) E(1) .(6) Q(1) T(1) 503 Q .(24) 0.07 41/5 3.21 73 N Q(23) 0.12 7/7 4.22 Q(70) N(69) H(3) .(4) L(1) T(1) 14 D S(23) 0.08 8/0 3.81 R(1) D(69) 489 E .(25) 0.12 26/19 2.65 .(6) E(64) T(1) H(1) 48 N N(83) 0.08 26/3 3.85 Q(2) S(3) S(2) G(8) K(1) continued in next column continued in next column

4 Table 2. continued Table 2. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) D(2) T(1) 17 L L(77) 0.14 16/4 3.82 S(1) I(9) 71 G S(24) 0.23 26/26 3.33 .(6) G(64) V(4) .(4) A(1) T(1) 69 D D(91) 0.14 1/1 4.75 R(1) .(4) E(1) M(1) A(2) S(2) 76 L L(76) 0.23 2/2 4.29 74 F F(91) 0.14 80/16 2.78 V(13) .(4) .(3) C(2) I(2) Y(1) S(1) 513 F .(25) 0.14 29/2 3.40 A(1) Y(43) F(1) F(28) 495 A .(25) 0.25 19/11 3.96 I(1) A(46) S(1) S(25) 502 A .(24) 0.15 2/1 4.34 H(1) A(46) S(27) Table 2. The top 25% of residues in 1tt5A at the interface with 1tt5B. P(1) (Field names: res: residue number in the PDB entry; type: amino acid type; 522 Y .(27) 0.15 2/0 4.86 substs: substitutions seen in the alignment; with the percentage of each type F(38) in the bracket; noc/bb: number of contacts with the ligand, with the number of Y(33) contacts realized through backbone atoms given in the bracket; dist: distance S(1) of closest apporach to the ligand. ) 158 L L(56) 0.16 26/2 3.48 F(34) Table 3. M(7) Y(1) res type disruptive 21 G G(70) 0.17 1/1 4.84 mutations A(20) 157 G (KER)(FQMWHD)(NYLPI)(SVA) .(4) 504 E (FWH)(VCAG)(YR)(T) K(1) 507 K (Y)(FTW)(SVCAG)(HD) N(1) 331 D (R)(FWH)(VCAG)(KY) X(1) 47 K (Y)(FW)(T)(VCAG) 515 I .(25) 0.18 7/7 3.98 484 R (D)(T)(LPI)(YE) P(60) 44 E (FW)(H)(Y)(VCAG) I(11) 332 M (Y)(T)(H)(SCRG) F(1) 499 G (KR)(E)(FMWH)(Q) K(1) 51 L (R)(Y)(T)(H) 493 I .(25) 0.19 3/1 4.23 96 L (YR)(T)(H)(KECG) I(39) 481 E (FWH)(R)(YVCAG)(TK) V(29) 13 Y (KM)(VA)(Q)(LPI) T(2) 15 R (T)(D)(YVCAG)(S) M(1) 16 Q (Y)(FWH)(T)(VCAG) P(1) 18 R (D)(T)(EVLAPI)(SCG) 52 P A(14) 0.22 7/4 3.92 20 W (KE)(T)(QD)(R) P(50) 211 H (T)(VCAG)(E)(S) G(29) 503 Q (Y)(T)(SFCWHG)(VA) .(2) 14 D (R)(FWH)(K)(YVQMA) continued in next column continued in next column

5 Table 3. continued Figure 5 shows residues in 1tt5A colored by their importance, at the res type disruptive interface with 1tt5B. mutations 48 N (Y)(H)(ER)(FW) 2.4.3 Possible novel functional surfaces at 25% coverage. One 334 A (KR)(E)(YQ)(H) group of residues is conserved on the 1tt5A surface, away from (or 336 S (KR)(FWH)(QM)(LPI) susbtantially larger than) other functional sites and interfaces reco- 343 Q (Y)(T)(FWH)(S) gnizable in PDB entry 1tt5. It is shown in Fig. 6. The residues 491 H (T)(E)(D)(S) 19 L (YR)(H)(T)(KE) 95 E (FWH)(Y)(CG)(VA) 339 Y (K)(QM)(E)(NVLAPI) 12 K (Y)(FW)(T)(SVCAG) 73 N (Y)(FW)(H)(T) 489 E (FW)(H)(Y)(VCAG) 17 L (R)(Y)(H)(T) 69 D (R)(H)(FW)(Y) 74 F (K)(E)(Q)(D) 513 F (K)(E)(Q)(R) 502 A (R)(KY)(E)(H) 522 Y (K)(Q)(M)(NER) 158 L (R)(TY)(K)(EH) 21 G (E)(R)(FWH)(K) 515 I (Y)(T)(R)(H) 493 I (R)(Y)(H)(T) 52 P (R)(Y)(H)(K) 71 G (KR)(E)(FWH)(M) 76 L (R)(Y)(H)(K) 495 A (KE)(R)(YQ)(D)

Table 3. List of disruptive mutations for the top 25% of residues in 1tt5A, that are at the interface with 1tt5B. Fig. 6. A possible active surface on the chain 1tt5A.

belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 4.6). Table 4. res type substitutions(%) cvg 97 N N(100) 0.00 504 E .(24)E(75) 0.01 507 K .(24)K(75) 0.01 47 K K(96)S(1).(2) 0.03 484 R .(25)R(71)S(2) 0.03 44 E E(92)R(1).(2) 0.04 Q(2)X(1) 499 G .(25)G(72)S(1) 0.04 51 L L(95).(2)I(1) 0.05 F(1) 96 L L(95)F(2)M(2) 0.05 100 V V(95)T(3)S(1) 0.06 481 E .(25)E(71)D(1) 0.06 S(1) 13 Y Y(92).(6)K(1) 0.07 15 R R(92).(6)N(1) 0.07 16 Q Q(92).(6)E(1) 0.07 continued in next column Fig. 5. Residues in 1tt5A, at the interface with 1tt5B, colored by their relative importance. 1tt5B is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1tt5A.)

6 Table 4. continued Table 4. continued res type substitutions(%) cvg res type substitutions(%) cvg 18 R Y(23)R(70).(4) 0.07 493 I .(25)I(39)V(29) 0.19 X(1) T(2)M(1)P(1) 20 W L(23)W(70).(4) 0.07 510 T .(27)T(64)S(4) 0.19 X(1) A(2)I(1) 25 Q M(23)Q(70).(4) 0.07 395 C .(25)A(2)G(34) 0.20 X(1) C(16)Y(14)F(1) 503 Q .(24)Q(70)H(3) 0.07 W(2)V(1)T(1) L(1) 52 P A(14)P(50)G(29) 0.22 14 D S(23)D(69).(6) 0.08 .(2)T(1)S(1) T(1) 71 G S(24)G(64).(4) 0.23 48 N N(83)S(3)G(8) 0.08 T(1)R(1)E(1) F(1).(2) A(2) 53 G G(93)C(1)P(1) 0.08 76 L L(76)V(13).(3) 0.23 .(2)N(1) I(2)S(1)A(1) 491 H .(25)H(69)N(1) 0.09 F(1) P(2)Q(1) 9 K D(24)K(44).(17) 0.24 512 Q .(25)Q(70)R(1) 0.10 S(3)H(2)R(3) K(1)P(1) P(2)A(1) 19 L V(23)L(55)I(14) 0.11 31 A S(39)A(50).(3) 0.24 .(4)X(1) C(2)T(2)X(1) 95 E E(91)Q(2).(2) 0.11 518 N .(27)N(55)G(11) 0.24 A(1)R(1)D(1) H(1)K(1)Q(1) 12 K L(23)K(56)R(12) 0.12 R(1)S(1) .(6)T(1) 495 A .(25)A(46)S(25) 0.25 73 N Q(23)N(69).(4) 0.12 H(1) T(1)R(1) 489 E .(25)E(64)H(1) 0.12 Table 4. Residues forming surface ”patch” in 1tt5A. Q(2)S(2)K(1) D(2) 506 I .(24)I(56)L(12) 0.12 Table 5. V(6) res type disruptive 17 L L(77)I(9).(6) 0.14 mutations V(4)A(1) 97 N (Y)(FTWH)(SEVCARG)(MD) 69 D D(91).(4)M(1) 0.14 504 E (FWH)(VCAG)(YR)(T) S(2) 507 K (Y)(FTW)(SVCAG)(HD) 74 F F(91).(4)C(2) 0.14 47 K (Y)(FW)(T)(VCAG) Y(1) 484 R (D)(T)(LPI)(YE) 513 F .(25)Y(43)F(28) 0.14 44 E (FW)(H)(Y)(VCAG) I(1)S(1) 499 G (KR)(E)(FMWH)(Q) 55 G K(23)G(66)R(2) 0.15 51 L (R)(Y)(T)(H) P(1).(2)Q(2) 96 L (YR)(T)(H)(KECG) A(1) 100 V (KR)(E)(Y)(QH) 502 A .(24)A(46)S(27) 0.15 481 E (FWH)(R)(YVCAG)(TK) P(1) 13 Y (KM)(VA)(Q)(LPI) 522 Y .(27)F(38)Y(33) 0.15 15 R (T)(D)(YVCAG)(S) S(1) 16 Q (Y)(FWH)(T)(VCAG) 158 L L(56)F(34)M(7) 0.16 18 R (D)(T)(EVLAPI)(SCG) Y(1) 20 W (KE)(T)(QD)(R) 21 G G(70)A(20).(4) 0.17 25 Q (Y)(TH)(FW)(SCG) K(1)N(1)X(1) 503 Q (Y)(T)(SFCWHG)(VA) 515 I .(25)P(60)I(11) 0.18 14 D (R)(FWH)(K)(YVQMA) F(1)K(1) 48 N (Y)(H)(ER)(FW) continued in next column 53 G (R)(E)(K)(H) continued in next column

7 Table 5. continued Table 6. res type disruptive res type substitutions(%) cvg mutations 264 N .(23)N(76) 0.01 491 H (T)(E)(D)(S) 267 E .(23)E(76) 0.01 512 Q (Y)(T)(FW)(H) 324 P .(25)P(74) 0.02 19 L (YR)(H)(T)(KE) 330 P .(25)P(74) 0.02 95 E (FWH)(Y)(CG)(VA) 331 D .(25)D(74) 0.02 12 K (Y)(FW)(T)(SVCAG) 353 D .(23)D(75)H(1) 0.02 73 N (Y)(FW)(H)(T) 225 W .(23)W(69)V(1) 0.04 489 E (FW)(H)(Y)(VCAG) C(2)Y(1)F(2) 506 I (YR)(H)(T)(KE) 350 A .(23)A(71)S(2) 0.05 17 L (R)(Y)(H)(T) P(1)V(1) 69 D (R)(H)(FW)(Y) 211 H .(24)H(64)K(8) 0.07 74 F (K)(E)(Q)(D) E(1)Q(1) 513 F (K)(E)(Q)(R) 327 G .(25)G(71)K(1) 0.08 55 G (E)(YDR)(FWH)(K) Y(1) 502 A (R)(KY)(E)(H) 384 C .(24)C(66)S(4) 0.08 522 Y (K)(Q)(M)(NER) V(2)I(1) 158 L (R)(TY)(K)(EH) 234 P .(25)P(69)L(1) 0.09 21 G (E)(R)(FWH)(K) F(1)S(1)E(1) 515 I (Y)(T)(R)(H) 334 A .(25)A(45)S(24) 0.09 493 I (R)(Y)(H)(T) T(1)C(2) 510 T (R)(K)(H)(Q) 336 S .(25)S(39)T(34) 0.09 395 C (K)(E)(R)(Q) 343 Q .(25)Q(66)A(3) 0.09 52 P (R)(Y)(H)(K) P(1)K(1)R(1) 71 G (KR)(E)(FWH)(M) 222 L .(24)L(54)A(18) 0.10 76 L (R)(Y)(H)(K) M(1)S(1) 9 K (Y)(T)(FW)(CG) 262 E .(23)E(64)Q(2) 0.11 31 A (KR)(E)(Y)(Q) S(7)A(1)T(1) 518 N (Y)(FW)(T)(H) 339 Y .(25)Y(65)F(7) 0.11 495 A (KE)(R)(YQ)(D) R(1) 209 H .(28)H(56)T(8) 0.12 Table 5. Disruptive mutations for the surface patch in 1tt5A. F(2)L(2)R(1) 240 K .(23)K(65)R(2) 0.13 C(1)V(4)D(1) Another group of surface residues is shown in Fig.7. The right panel T(1) shows (in blue) the rest of the larger cluster this surface belongs to. 263 E .(23)E(59)A(1) 0.13 D(11)T(1)G(2) M(1) 349 K .(23)K(59)Q(12) 0.13 L(1)R(2)E(1) 323 L .(25)L(60)A(6) 0.14 P(3)T(1)V(1) F(1) 184 R G(14)R(74).(4) 0.15 V(2)S(1)N(1) P(1) 385 S .(24)K(55)R(12) 0.15 S(6)E(1) 265 F .(23)F(43)Y(29) 0.16 Fig. 7. Another possible active surface on the chain 1tt5A. The larger cluster I(1)H(1)V(1) it belongs to is shown in blue. 325 V .(25)V(17)L(56) 0.16 continued in next column The residues belonging to this surface ”patch” are listed in Table 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 4.6).

8 Table 6. continued Table 7. res type substitutions(%) cvg res type disruptive 386 N .(24)N(59)K(1) 0.16 mutations E(8)H(6) 264 N (Y)(FTWH)(SVCAG)(ER) 210 S .(24)G(19)A(2) 0.17 267 E (FWH)(VCAG)(YR)(T) S(28)K(18)N(2) 324 P (YR)(TH)(SCG)(KE) R(2)D(1) 330 P (YR)(TH)(SCG)(KE) 214 W .(24)Y(45)W(20) 0.17 331 D (R)(FWH)(VCAG)(KY) F(7)M(1) 353 D (R)(FW)(VCAG)(H) 281 I .(23)L(18)I(37) 0.17 225 W (K)(E)(Q)(D) V(13)P(6)W(1) 350 A (R)(K)(Y)(E) 220 K .(24)H(22)K(45) 0.18 211 H (T)(VCAG)(E)(S) R(6)S(1) 327 G (E)(KR)(FMWD)(QH) 271 N .(23)A(50)S(7) 0.18 384 C (R)(K)(E)(H) N(18) 234 P (R)(Y)(H)(T) 375 I .(23)V(7)L(2) 0.19 334 A (KR)(E)(YQ)(H) I(64)N(1)A(1) 336 S (KR)(FWH)(QM)(LPI) 217 I .(24)I(30)V(13) 0.20 343 Q (Y)(T)(FWH)(S) L(30) 222 L (YR)(H)(T)(K) 326 R .(25)P(30)R(17) 0.20 262 E (H)(FW)(R)(Y) E(14)Q(1)S(4) 339 Y (K)(QM)(E)(NVLAPI) G(1)D(2)H(1) 209 H (E)(D)(T)(Q) 212 T .(24)L(3)V(29) 0.21 240 K (Y)(FW)(T)(VAH) T(24)I(17) 263 E (H)(FW)(R)(Y) 274 T .(23)K(37)R(8) 0.21 349 K (Y)(T)(FW)(CG) T(17)M(1)S(3) 323 L (R)(Y)(H)(K) Y(2)Q(1)I(3) 184 R (D)(Y)(T)(E) H(1) 385 S (FW)(H)(YR)(K) 380 L .(23)V(17)I(44) 0.21 265 F (K)(E)(Q)(D) C(3)L(7)A(2) 325 V (Y)(R)(KE)(H) F(1) 386 N (Y)(T)(FW)(VAH) 387 S .(24)A(54)S(11) 0.21 210 S (R)(FW)(KH)(Y) C(6)I(1)T(2) 214 W (K)(E)(TQD)(NCRG) 272 V .(23)V(45)I(3) 0.22 281 I (R)(Y)(T)(H) A(11)M(2)C(1) 220 K (Y)(T)(FW)(VCAG) S(8)L(3) 271 N (Y)(H)(FW)(R) 188 P A(2)P(75).(6) 0.23 375 I (Y)(R)(H)(T) C(11)Q(1)S(1) 217 I (YR)(H)(T)(KE) L(1)T(1) 326 R (T)(Y)(D)(CG) 261 D .(23)P(2)L(1) 0.23 212 T (R)(K)(H)(Q) D(43)E(6)R(1) 274 T (R)(K)(FW)(H) G(18)S(1)Y(2) 380 L (R)(Y)(H)(KE) 237 Y .(27)Y(44)R(18) 0.24 387 S (R)(K)(H)(Q) N(1)A(1)K(1) 272 V (R)(Y)(K)(E) F(3)T(1)S(1) 188 P (R)(Y)(H)(K) 266 E .(23)E(22)I(1) 0.25 261 D (R)(H)(FW)(K) D(30)K(13)V(2) 237 Y (K)(M)(Q)(E) Y(1)T(3)Q(1) 266 E (H)(FW)(R)(Y) 388 A .(24)A(39)Q(3) 0.25 388 A (Y)(R)(KE)(H) R(20)L(1)S(3) G(1)D(2)F(1) Table 7. Disruptive mutations for the surface patch in 1tt5A. P(1)

Table 6. Residues forming surface ”patch” in 1tt5A.

3 CHAIN 1TT5B 3.1 Q8TBC4 overview From SwissProt, id Q8TBC4, 96% identical to 1tt5B:

9 Description: Ubiquitin-activating enzyme E1C (Nedd8-activating enzyme E1C) (Ubiquitin-activating enzyme 3 homolog). Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini; Hominidae; Homo. Function: The dimeric enzyme activates NEDD8 by first adenylating its C-terminal glycine residue with ATP and thereafter linking this residue to the side chain of a cysteine residue on UBE1C, yielding a NEDD8-UBE1C thiolester and free AMP. Down-regulates steroid receptor activity. Necessary for cell cycle progression. Overexpres- sion of APPBP1 causes apoptosis through deregulation of NEDD8 conjugation (By similarity). Enzyme regulation: Binding of TP53BP2 to the regulatory subunit Fig. 8. Residues 9-215 in 1tt5B colored by their relative importance. (See APPBP1 decreases activity. Appendix, Fig.17, for the coloring scheme.) Pathway: NEDD8 conjugation; first step. Subunit: Heterodimer of UBE1C and APPBP1. The complex binds NEDD8. Binds ESR1 and ESR2 with bound steroid ligand (By similarity). Tissue specificity: Ubiquitous. Miscellaneous: APPBP1 and UBE1C correspond to the N-terminal and the C-terminal part of yeast UBA3. In yeast the two subunits form a single polypeptide chain. Similarity: Belongs to the ubiquitin-activating E1 family. Caution: It is uncertain whether Met-1 or Met-22 is the initiator. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. Fig. 9. Residues 216-1008 in 1tt5B colored by their relative importance. (See 3.2 Multiple sequence alignment for 1tt5B Appendix, Fig.17, for the coloring scheme.) For the chain 1tt5B, the alignment 1tt5B.msf (attached) with 269 sequences was used. The alignment was downloaded from the HSSP 3.3 Residue ranking in 1tt5B database, and fragments shorter than 75% of the query as well as The 1tt5B sequence is shown in Figs. 8–9, with each residue colored duplicate sequences were removed. It can be found in the attachment according to its estimated importance. The full listing of residues to this report, under the name of 1tt5B.msf. Its statistics, from the in 1tt5B can be found in the file called 1tt5B.ranks sorted in the alistat program are the following: attachment. Format: MSF 3.4 Top ranking residues in 1tt5B and their position on Number of sequences: 269 the structure Total number of residues: 77389 In the following we consider residues ranking among top 25% of Smallest: 86 residues in the protein . Figure 10 shows residues in 1tt5B colored Largest: 414 by their importance: bright red and yellow indicate more conser- Average length: 287.7 ved/important residues (see Appendix for the coloring scheme). A Alignment length: 414 Pymol script for producing this figure can be found in the attachment. Average identity: 34% Most related pair: 99% Most unrelated pair: 0% 3.4.1 Clustering of residues at 25% coverage. Fig. 11 shows the Most distant seq: 34% top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.11 are composed of the residues listed in Table 8. Furthermore, <1% of residues show as conserved in this ali- Table 8. gnment. cluster size member The alignment consists of 27% eukaryotic ( 4% vertebrata, 2% color residues arthropoda, 11% fungi, 1% plantae), 1% prokaryotic, and 1% red 99 52,55,56,57,58,59,60,61,62 archaean sequences. (Descriptions of some sequences were not rea- dily available.) The file containing the sequence descriptions can be continued in next column found in the attachment, under the name 1tt5B.descr.

10 Table 8. continued cluster size member color residues 138,141,143,144,145,146,147 150,151,153,155,158,176,177 178,179,180,181,182,184,185 187,188,190,197,198,199,200 202,214,216,217,218,219,221 222,223,225,226,227,228,229 230,231,232,233,250,252,259 260,264,267,268,271,273,284 286,288,290,291,293,294,295 296,297

Table 8. Clusters of top ranking residues in 1tt5B.

3.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with the peptide 1tt5E. Table 9 lists the top 25% of residues at the interface with 1tt5E. The following table (Table 10) Fig. 10. Residues in 1tt5B, colored by their relative importance. Clockwise: suggests possible disruptive replacements for these residues (see front, back, top and bottom views. Section 4.6). Table 9. res type subst’s cvg noc/ dist (%) bb (A˚ ) 176 P I(2) 0.03 7/0 3.81 P(90) .(2) L(1)RST AEY 138 F F(49) 0.15 9/9 3.24 S(1) Y(11) Q(2) L(23) A(3) I(1) V(2) H(1) C(1)K .(1) 197 T F(1) 0.24 3/0 3.97 T(77) Y(2) Fig. 11. Residues in 1tt5B, colored according to the cluster they belong to: S(7) red, followed by blue and yellow are the largest clusters (see Appendix for G(6) the coloring scheme). Clockwise: front, back, top and bottom views. The A(1)NCL corresponding Pymol script is attached. .(1)

Table 8. continued Table 9. The top 25% of residues in 1tt5B at the interface with 1tt5E. cluster size member (Field names: res: residue number in the PDB entry; type: amino acid type; color residues substs: substitutions seen in the alignment; with the percentage of each type 64,65,66,67,71,79,81,83,84 in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance 86,87,88,89,90,91,92,93,94 of closest apporach to the ligand. ) 98,99,100,103,104,107,134 continued in next column

11 Table 10. res type disruptive mutations 176 P (R)(Y)(H)(K) 138 F (E)(K)(D)(TQ) 197 T (KR)(Q)(H)(M)

Table 10. List of disruptive mutations for the top 25% of residues in 1tt5B, that are at the interface with 1tt5E.

Fig. 12. Residues in 1tt5B, at the interface with 1tt5E, colored by their rela- tive importance. 1tt5E is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1tt5B.)

Figure 12 shows residues in 1tt5B colored by their importance, at the interface with 1tt5E. Interface with 1tt5D.Table 11 lists the top 25% of residues at the interface with 1tt5D. The following table (Table 12) suggests possible disruptive replacements for these residues (see Section 4.6). Table 11. res type subst’s cvg noc/ dist (%) bb (A˚ ) 134 F F(63) 0.19 35/9 3.37 W(8) D(1) L(8) Y(8) I(4) V(1) H(1) .(1)TA continued in next column

12 Table 11. continued Figure 13 shows residues in 1tt5B colored by their importance, at the res type subst’s cvg noc/ dist interface with 1tt5D. (%) bb (A˚ ) Zinc ion binding site. Table 13 lists the top 25% of residues at 153 W F(2) 0.23 1/0 4.91 the interface with 1tt5ZN301 (zinc ion). The following table (Table H(18) 14) suggests possible disruptive replacements for these residues (see I(1) Section 4.6). Y(38) W(21) Table 13. L(10) res type subst’s cvg noc/ dist ˚ V(1) (%) bb (A) M(1) 199 C C(66) 0.03 6/4 2.56 A(1) S(26) .(1)CTK N(2)T Q A(1)R .(1)M 202 C C(65) 0.07 4/2 2.44 Table 11. The top 25% of residues in 1tt5B at the interface with 1tt5D. (Field names: res: residue number in the PDB entry; type: amino acid type; D(1) substs: substitutions seen in the alignment; with the percentage of each type S(24) in the bracket; noc/bb: number of contacts with the ligand, with the number of A(2) contacts realized through backbone atoms given in the bracket; dist: distance E(1) of closest apporach to the ligand. ) G(1) .(2)P

Table 12. Table 13. The top 25% of residues in 1tt5B at the interface with zinc res type disruptive ion.(Field names: res: residue number in the PDB entry; type: amino acid mutations type; substs: substitutions seen in the alignment; with the percentage of each 134 F (K)(E)(Q)(R) type in the bracket; noc/bb: number of contacts with the ligand, with the num- 153 W (E)(K)(D)(T) ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 12. List of disruptive mutations for the top 25% of residues in 1tt5B, that are at the interface with 1tt5D. Table 14. res type disruptive mutations 199 C (ER)(K)(H)(FW) 202 C (R)(K)(H)(FEW)

Table 14. List of disruptive mutations for the top 25% of residues in 1tt5B, that are at the interface with zinc ion.

Figure 14 shows residues in 1tt5B colored by their importance, at the interface with 1tt5ZN301. Interface with 1tt5A.Table 15 lists the top 25% of residues at the interface with 1tt5A. The following table (Table 16) suggests possible disruptive replacements for these residues (see Section 4.6). Table 15. res type subst’s cvg noc/ dist (%) bb (A˚ ) 90 R R(98)M. 0.01 22/11 3.35 S 88 L I(2) 0.04 12/9 4.10 L(96). 222 P P(82) 0.04 17/12 2.48 .(16)GS L 227 H H(80) 0.04 5/0 3.78 Fig. 13. Residues in 1tt5B, at the interface with 1tt5D, colored by their rela- continued in next column tive importance. 1tt5D is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1tt5B.)

13 Table 15. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) P(13) V(1)L .(2)WYS 86 S S(85) 0.09 35/19 3.26 T(10) A(1)P .(1)H 89 N S(9) 0.10 71/31 2.99 N(76) Q(10) H(1)VP. 92 F F(81) 0.11 37/7 3.38 I(10) L(1) V(4)C .(1) 290 P P(81)K 0.12 35/16 3.19 H(2) .(5) Fig. 14. Residues in 1tt5B, at the interface with zinc ion, colored by their G(8)QSA relative importance. The ligand (zinc ion) is colored green. Atoms further L than 30A˚ away from the geometric center of the ligand, as well as on the line 271 Y Y(34) 0.14 12/4 3.80 of sight to the ligand were removed. (See Appendix for the coloring scheme F(42) for the protein chain 1tt5B.) .(18) N(1)ACS Table 15. continued LHK res type subst’s cvg noc/ dist 293 A I(1) 0.15 15/5 3.85 (%) bb (A˚ ) A(68) .(17)E G(12) D(1)GN S(3)N 184 G G(87) 0.06 4/4 4.07 P(2) Q(2) .(4)T R(7)EAD V(2)D K. L(1)CEQ 214 P P(84) 0.06 1/0 4.98 K .(7) 66 N N(59) 0.16 23/6 3.56 A(5) Y(15) Q(1)NLE S(1) 93 L F(2) 0.07 2/0 3.87 L(1) L(88) T(2) I(5)S.A D(8)M YV .(2) 217 T S(3) 0.07 5/0 3.06 V(4)Q T(80) C(1)FIA .(7) 223 R T(4) 0.17 106/12 2.58 A(6)VQ S(21) E(1)I .(16) 273 I I(74) 0.08 4/0 3.03 Y(2) .(18)S R(25) L(2) A(1) M(2)A N(20) V(1)FR K(1) 62 E E(80)IA 0.09 13/0 3.12 continued in next column continued in next column

14 Table 15. continued Table 15. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) H(2) N(1) E(1) V(1) D(1)VP R(2)KP 297 A MA(67) 0.17 15/8 3.85 A(1)GHM G(15)C 288 I T(2) 0.22 37/1 3.62 .(4) I(76) S(8) K(1) T(1)IDF V(6) 291 A T(2) 0.18 36/33 2.86 .(10)DE A(73) LS V(10) 185 F L(5) 0.23 52/0 3.21 E(1) F(41) .(5)N Y(16) S(1)DI T(27)S G(1)KYP P(2) 65 K K(77) 0.20 60/5 2.82 W(4)IQA Q(2) . H(1) 220 S GS(31) 0.25 2/2 4.30 .(2)I .(15) Y(1)S Q(1) L(5) H(4) R(1) N(31) M(1) E(2) T(2)AEF T(6) C A(2)MDF 221 M R(2) 0.21 58/16 3.20 RY T(32) .(15) Table 15. The top 25% of residues in 1tt5B at the interface with 1tt5A. F(30) (Field names: res: residue number in the PDB entry; type: amino acid type; M(2) substs: substitutions seen in the alignment; with the percentage of each type V(1) in the bracket; noc/bb: number of contacts with the ligand, with the number of I(7) contacts realized through backbone atoms given in the bracket; dist: distance N(3)LCS of closest apporach to the ligand. ) Q 286 K Q(1) 0.21 2/0 4.47 Table 16. G(49) K(28) res type disruptive .(14)TE mutations HC(1) 90 R (TD)(Y)(CG)(SEVLAPI) A(1) 88 L (Y)(R)(T)(H) L(1)DYN 222 P (R)(Y)(H)(K) 294 S S(28) 0.21 39/19 3.28 227 H (E)(T)(M)(Q) T(48) 184 G (FW)(HR)(YE)(K) F(1) 214 P (Y)(R)(H)(T) .(4) 93 L (R)(Y)(K)(H) V(4) 217 T (R)(K)(H)(FW) P(6) 273 I (Y)(R)(T)(H) A(3) 62 E (R)(H)(Y)(FW) G(1)YI 86 S (KR)(Q)(MH)(FW) 306 E E(77) 0.21 15/1 2.56 89 N (Y)(H)(T)(FW) .(4) 92 F (KE)(QR)(D)(T) Q(7)ID 290 P (Y)(R)(H)(T) 271 Y (K)(Q)(EM)(R) continued in next column continued in next column

15 Table 16. continued res type disruptive mutations 293 A (Y)(R)(H)(K) 66 N (Y)(H)(R)(FW) 223 R (T)(D)(Y)(E) 297 A (R)(K)(E)(Y) 291 A (R)(Y)(K)(EH) 65 K (Y)(T)(FW)(CG) 221 M (Y)(H)(T)(R) 286 K (Y)(FW)(T)(VA) 294 S (R)(K)(Q)(H) 306 E (H)(Y)(FW)(R) Fig. 16. A possible active surface on the chain 1tt5B. The larger cluster it belongs to is shown in blue. 288 I (R)(Y)(H)(T) 185 F (K)(E)(QR)(D) 220 S (R)(K)(H)(FW)

Table 16. List of disruptive mutations for the top 25% of residues in 1tt5B, that are at the interface with 1tt5A.

Fig. 15. Residues in 1tt5B, at the interface with 1tt5A, colored by their rela- tive importance. 1tt5A is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1tt5B.)

Figure 15 shows residues in 1tt5B colored by their importance, at the interface with 1tt5A. 3.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1tt5B surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1tt5. It is shown in Fig. 16. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 17, while Table 18 suggests possible disruptive replacements for these residues (see Section 4.6).

16 Table 17. Table 17. continued res type substitutions(%) cvg res type substitutions(%) cvg 146 D D(98).(1) 0.00 T(2)N.H 81 D D(97)Q(1).(1)G 0.01 188 N S(2)Q(49)N(34) 0.10 87 N N(97)D(1).(1) 0.01 H(11)YLI.R 90 R R(98)M.S 0.01 92 F F(81)I(10)L(1) 0.11 91 Q Q(98)T. 0.01 V(4)C.(1) 103 K K(98)P.R 0.01 182 T V(6)T(74)I(8) 0.11 79 D D(97).(1)GE 0.02 S(6)L(1)CA(1). 151 R R(96)K(2).(1) 0.02 231 Y H(1)W(61).(17) 0.11 216 C C(91).(7)Q 0.02 Y(18)F(1)LVS 181 G G(86)A(9)S(4). 0.03 84 D E(49)D(45)T(1) 0.12 88 L I(2)L(96). 0.04 S(1).(1)QV 147 S N(73)S(24)A(1) 0.04 290 P P(81)KH(2).(5) 0.12 .(1) G(8)QSAL 222 P P(82).(16)GSL 0.04 98 D D(66)H(22)N(5) 0.13 227 H H(80).(17)ED(1) 0.04 .(1)S(1)RT(1)LE GN 225 P F(2)P(43).(17) 0.14 94 F F(82)H(12)Y(2)K 0.05 I(27)A(2)V(1)S .SL M(3)H(1)LTC 187 G G(91)A(2)C(5)D. 0.05 250 D N(4)D(67).(18) 0.14 57 G G(95)D.(3)R 0.06 S(1)E(4)K(1)Y 144 G C(4)A(59)G(34) 0.06 R(1)AP .(1) 271 Y Y(34)F(42).(18) 0.14 184 G G(87)Q(2)R(7)EA 0.06 N(1)ACSLHK DK. 293 A I(1)A(68)G(12) 0.15 214 P P(84).(7)A(5) 0.06 S(3)NP(2).(4)T Q(1)NLE V(2)DL(1)CEQK 58 G G(64)A(31)W.(3) 0.07 66 N N(59)Y(15)S(1) 0.16 83 I V(19)I(78)P.(1) 0.07 L(1)T(2)D(8)M FAL .(2)V(4)QC(1)FI 93 L F(2)L(88)I(5)S. 0.07 A AYV 252 D S(1)D(71).(18)Q 0.16 217 T S(3)T(80).(7) 0.07 N(2)HA(1)E(2)FM A(6)VQE(1)I PK 145 L L(84)V(2)M(1) 0.08 268 A A(60)S(9).(17) 0.16 T(8)S(2).(1)C M(3)EQ(1)C(3)P 273 I I(74).(18)SL(2) 0.08 G(1)DVLT M(2)AV(1)FR 223 R T(4)S(21).(16) 0.17 62 E E(80)IAP(13) 0.09 Y(2)R(25)A(1) V(1)L.(2)WYS N(20)K(1)H(2) 86 S S(85)T(10)A(1)P 0.09 E(1)D(1)VP .(1)H 297 A MA(67)G(15)C 0.17 150 A S(3)A(78)T(14)G 0.09 .(4)S(8)T(1)IDF VDP(1).(1) 226 E TI(15).(17) 0.18 267 R R(75).(17)E(1) 0.09 E(42)V(7)N(2) H(1)QSYMVFLI D(6)A(1)Q(5)FK 59 L L(46)I(49)V.(2) 0.10 291 A T(2)A(73)V(10) 0.18 89 N S(9)N(76)Q(10) 0.10 E(1).(5)NS(1)DI H(1)VP. G(1)KYP 100 G G(76)K(7)Q(5) 0.10 296 N L(2)N(47)A(2)F 0.18 D(2)R(1)ES(1) T(26).(4)P(8) continued in next column S(2)CV(2)K(1)QI 219 A A(28)R(28).(8) 0.19 continued in next column

17 Table 17. continued Table 18. continued res type substitutions(%) cvg res type disruptive K(19)T(2)H(5)EI mutations WG(2)V 216 C (R)(E)(FWH)(K) 65 K K(77)Q(2)H(1) 0.20 181 G (KR)(E)(QH)(FMWD) .(2)IY(1)SL(5) 88 L (Y)(R)(T)(H) R(1)M(1)T(2)AEF 147 S (R)(K)(H)(FW) C 222 P (R)(Y)(H)(K) 233 R M(1)K(32).(17) 0.20 227 H (E)(T)(M)(Q) R(28)Q(1)H(7) 94 F (E)(K)(T)(D) L(1)S(5)Y(1)TV 187 G (R)(K)(E)(H) N(1)I 57 G (FKEWR)(MH)(QD)(YLPI) 221 M R(2)T(32).(15) 0.21 144 G (KER)(QHD)(FMW)(NY) F(30)M(2)V(1) 184 G (FW)(HR)(YE)(K) I(7)N(3)LCSQ 214 P (Y)(R)(H)(T) 286 K Q(1)G(49)K(28) 0.21 58 G (KE)(R)(QD)(MH) .(14)TEHC(1) 83 I (R)(Y)(T)(H) A(1)L(1)DYN 93 L (R)(Y)(K)(H) 294 S S(28)T(48)F(1) 0.21 217 T (R)(K)(H)(FW) .(4)V(4)P(6) 145 L (R)(Y)(H)(K) A(3)G(1)YI 273 I (Y)(R)(T)(H) 99 I I(34)L(4)V(58)P 0.22 62 E (R)(H)(Y)(FW) AE.R 86 S (KR)(Q)(MH)(FW) 288 I T(2)I(76)K(1) 0.22 150 A (R)(K)(Y)(E) V(6).(10)DELS 267 R (T)(D)(Y)(CG) 153 W F(2)H(18)I(1) 0.23 59 L (YR)(H)(T)(KE) Y(38)W(21)L(10) 89 N (Y)(H)(T)(FW) V(1)M(1)A(1) 100 G (FW)(R)(E)(H) .(1)CTKQ 188 N (Y)(T)(FWH)(E) 185 F L(5)F(41)Y(16) 0.23 92 F (KE)(QR)(D)(T) T(27)SP(2)W(4)I 182 T (R)(K)(H)(Q) QA. 231 Y (K)(Q)(E)(M) 284 V I(36)M(12)ET 0.24 84 D (R)(H)(FW)(Y) V(29).(15)K(1) 290 P (Y)(R)(H)(T) L(1)SG(1) 98 D (R)(FW)(H)(Y) 148 I I(25)L(13)F(14) 0.25 225 P (R)(Y)(H)(K) V(35)YNR(5)WK 250 D (R)(FWH)(Y)(CG) .(1)MQA 271 Y (K)(Q)(EM)(R) 220 S GS(31).(15)Q(1) 0.25 293 A (Y)(R)(H)(K) H(4)N(31)E(2) 66 N (Y)(H)(R)(FW) T(6)A(2)MDFRY 252 D (R)(H)(FYW)(CG) 268 A (R)(Y)(K)(H) Table 17. Residues forming surface ”patch” in 1tt5B. 223 R (T)(D)(Y)(E) 297 A (R)(K)(E)(Y) 226 E (H)(FW)(Y)(R) Table 18. 291 A (R)(Y)(K)(EH) res type disruptive 296 N (Y)(H)(FW)(R) mutations 219 A (E)(Y)(KR)(D) 146 D (R)(FWH)(VCAG)(KY) 65 K (Y)(T)(FW)(CG) 81 D (R)(FWH)(Y)(VA) 233 R (T)(D)(Y)(E) 87 N (Y)(FWH)(T)(VCARG) 221 M (Y)(H)(T)(R) 90 R (TD)(Y)(CG)(SEVLAPI) 286 K (Y)(FW)(T)(VA) 91 Q (Y)(FWH)(TVA)(S) 294 S (R)(K)(Q)(H) 103 K (Y)(T)(FW)(SCG) 99 I (Y)(R)(H)(T) 79 D (R)(FWH)(KYVA)(CG) 288 I (R)(Y)(H)(T) 151 R (T)(D)(Y)(VCAG) continued in next column continued in next column

18 Table 18. continued backbone atoms (if all or most contacts are through the backbone, res type disruptive mutation presumably won’t have strong impact). Two heavy atoms mutations are considered to be “in contact” if their centers are closer than 5A˚ . 153 W (E)(K)(D)(T) 185 F (K)(E)(QR)(D) 4.5 Annotation 284 V (Y)(R)(KH)(E) If the residue annotation is available (either from the pdb file or 148 I (Y)(R)(T)(H) from other sources), another column, with the header “annotation” 220 S (R)(K)(H)(FW) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 18. Disruptive mutations for the surface patch in 1tt5B. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 4.6 Mutation suggestions 4 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- 4.1 Coverage mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less 4.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For 5 APPENDIX example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- 5.1 File formats sely, when looking for substitutions which will not affect the protein, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment • is given in the immediately following bracket. No percentage is given alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • residue# residue number in the PDB file guide - due to rounding errors these percentages often do not add up • type amino acid type to 100%. • rank rank of the position according to older version of ET 4.3 Surface • variability has two subfields: To detect candidates for novel functional interfaces, first we look for 1. number of different amino acids appearing in in this column residues that are solvent accessible (according to DSSP program) by of the alignment 2 at least 10A˚ , which is roughly the area needed for one water mole- 2. their type cule to come in the contact with the residue. Furthermore, we require • rho ET score - the smaller this value, the lesser variability of that these residues form a “cluster” of residues which have neighbor this position across the branches of the tree (and, presumably, within 5A˚ from any of their heavy atoms. the greater the importance for the protein) Note, however, that, if our picture of protein evolution is correct, • the neighboring residues which are not surface accessible might be cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller should not be automatically dropped from consideration when choo- • gaps percentage of gaps in this column sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 5.2 Color schemes used The following color scheme is used in figures with residues colored 4.4 Number of contacts by cluster size: black is a single-residue cluster; clusters composed of Another column worth noting is denoted “noc/bb”; it tells the num- more than one residue colored according to this hierarchy (ordered ber of contacts heavy atoms of the residue in question make across by descending size): red, blue, yellow, green, purple, azure, tur- the interface, as well as how many of them are realized through the quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold,

19 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997.

http://swift.cmbi.kun.nl/swift/hssp/ COVERAGE 5.3.5 LaTex The text for this report was processed using LATEX; V Leslie Lamport, “LaTeX: A Document Preparation System Addison- 100% 50% 30% 5% Wesley,” Reading, Mass. (1986).

5.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. V http://www.drive5.com/muscle/ RELATIVE IMPORTANCE 5.3.7 Pymol The figures in this report were produced using Fig. 17. Coloring scheme used to color residues by their relative importance. Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- tific LLC (2005). For more information about Pymol see bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, http://pymol.sourceforge.net/. (Note for Windows DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, users: the attached package needs to be unzipped for Pymol to read tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. the scripts and launch the viewer.) The colors used to distinguish the residues by the estimated 5.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 17. Dan Morgan from the Lichtarge lab has developed a visualization 5.3 Credits tool specifically for viewing trace results. If you are interested, please 5.3.1 Alistat alistat reads a multiple sequence alignment from the visit: file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two 5.5 Citing this work sequences. The ”average percent identity”, ”most related pair”, and The method used to rank residues and make predictions in this report ”most unrelated pair” of the alignment are the average, maximum, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant Evolution-Entropy Hybrid Methods for Ranking of Protein Residues seq” is calculated by finding the maximum pairwise identity (best by Importance” J. Mol. Bio. 336: 1265-82. For the original version relative) for all N sequences, then finding the minimum of these N of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- numbers (hence, the most outlying sequence). alistat is copyrighted tionary Trace Method Defines Binding Surfaces Common to Protein by HHMI/Washington University School of Medicine, 1992-2001, Families” J. Mol. Bio. 257: 342-358. and freely distributed under the GNU General Public License. report maker itself is described in Mihalek I., I. Res and O. 5.3.2 CE To map ligand binding sites from different Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type source structures, report maker uses the CE program: of service for comparative analysis of proteins.” Bioinformatics http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 22:1656-7. ”Protein structure alignment by incremental combinatorial extension 5.6 About report maker (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- 5.3.3 DSSP In this work a residue is considered solvent accessi- king visualization program was written by Ivica Res.ˇ report maker 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , is copyrighted by Lichtarge Lab, Baylor College of Medicine, which is roughly the area needed for one water molecule to come in Houston. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 5.7 Attachments by [email protected] November 18,2002, The following files should accompany this report: http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1tt5A.complex.pdb - coordinates of 1tt5A with all of its interac- 5.3.4 HSSP Whenever available, report maker uses HSSP ali- ting partners gnment as a starting point for the analysis (sequences shorter than • 1tt5A.etvx - ET viewer input file for 1tt5A

20 • 1tt5A.cluster report.summary - Cluster report summary for • 1tt5B.cluster report.summary - Cluster report summary for 1tt5A 1tt5B • 1tt5A.ranks - Ranks file in sequence order for 1tt5A • 1tt5B.ranks - Ranks file in sequence order for 1tt5B • 1tt5A.clusters - Cluster descriptions for 1tt5A • 1tt5B.clusters - Cluster descriptions for 1tt5B • 1tt5A.msf - the multiple sequence alignment used for the chain • 1tt5B.msf - the multiple sequence alignment used for the chain 1tt5A 1tt5B • 1tt5A.descr - description of sequences used in 1tt5A msf • 1tt5B.descr - description of sequences used in 1tt5B msf • 1tt5A.ranks sorted - full listing of residues and their ranking for • 1tt5B.ranks sorted - full listing of residues and their ranking for 1tt5A 1tt5B • 1tt5A.1tt5B.if.pml - Pymol script for Figure 5 • 1tt5B.1tt5E.if.pml - Pymol script for Figure 12 • 1tt5A.cbcvg - used by other 1tt5A – related pymol scripts • 1tt5B.cbcvg - used by other 1tt5B – related pymol scripts • 1tt5B.complex.pdb - coordinates of 1tt5B with all of its interac- • 1tt5B.1tt5D.if.pml - Pymol script for Figure 13 ting partners • 1tt5B.1tt5ZN301.if.pml - Pymol script for Figure 14 • 1tt5B.etvx - ET viewer input file for 1tt5B • 1tt5B.1tt5A.if.pml - Pymol script for Figure 15

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