Pages 1–13 2wqz Evolutionary trace report by report maker July 4, 2010

4 Notes on using trace results 11 4.1 Coverage 11 4.2 Known substitutions 11 4.3 Surface 11 4.4 Number of contacts 11 4.5 Annotation 11 4.6 Mutation suggestions 11

5 Appendix 11 5.1 File formats 11 5.2 Color schemes used 12 5.3 Credits 12 5.3.1 Alistat 12 5.3.2 CE 12 5.3.3 DSSP 12 5.3.4 HSSP 12 5.3.5 LaTex 12 5.3.6 Muscle 12 5.3.7 Pymol 12 5.4 Note about ET Viewer 12 5.5 Citing this work 12 5.6 About report maker 13 CONTENTS 5.7 Attachments 13

1 Introduction 1 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2wqz): 2 Chain 2wqzA 1 Title: Structural analysis of the synaptic protein neuroligin and its 2.1 Q8N0W4 overview 1 beta-neurexin complex: determinants for folding and cell adhesion 2.2 Multiple sequence alignment for 2wqzA 1 Compound: Mol id: 1; molecule: neuroligin 4, x-linked; chain: a, 2.3 Residue ranking in 2wqzA 2 b; fragment: acetylcholinesterase-like domain, residues 43-619; syn- 2.4 Top ranking residues in 2wqzA and their position on onym: neuroligin x, hnlx, neuroligin 4; engineered: yes; mol id: 2; the structure 2 molecule: neurexin-1-beta; chain: c, d; fragment: lns domain, resi- 2.4.1 Clustering of residues at 25% coverage. 2 dues 80-258; synonym: neurexin i-beta, beta-neurexin 1; engineered: 2.4.2 Overlap with known functional surfaces at yes 25% coverage. 3 Organism, scientific name: Rattus Norvegicus; 2.4.3 Possible novel functional surfaces at 25% 2wqz contains unique chains 2wqzA (545 residues) and 2wqzC coverage. 4 (177 residues) 2wqzB is a homologue of chain 2wqzA. 2wqzD is a homologue of chain 2wqzC. 3 Chain 2wqzC 7 3.1 P58400 overview 7 2 CHAIN 2WQZA 3.2 Multiple sequence alignment for 2wqzC 7 3.3 Residue ranking in 2wqzC 7 2.1 Q8N0W4 overview 3.4 Top ranking residues in 2wqzC and their position on From SwissProt, id Q8N0W4, 94% identical to 2wqzA: the structure 7 Description: Neuroligin 4, X linked precursor (Neuroligin X) 3.4.1 Clustering of residues at 25% coverage. 8 (HNLX). 3.4.2 Overlap with known functional surfaces at Organism, scientific name: Homo sapiens (Human). 25% coverage. 8 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.4.3 Possible novel functional surfaces at 25% Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; coverage. 9 Catarrhini; Hominidae; Homo.

1 Lichtarge lab 2006 Function: Putative neuronal cell surface protein involved in cell- cell-interactions. Subunit: Interacts through its C-terminus with DLG4/PSD-95 third PDZ domain. Subcellular location: Type I membrane protein (Potential). Alternative products: Event=Alternative splicing; Named isoforms=2; Name=1; IsoId=Q8N0W4-1; Sequence=Displayed; Name=2; IsoId=Q8N0W4-2; Sequence=VSP 013270; Note=No experimental confirmation available; Tissue specificity: Expressed at highest levels in heart. Expressed at lower levels in liver, skeletal muscle and pancreas and at very low levels in brain. Disease: Defects in NLGN4X may be the cause of susceptibility to X-linked autism 2 (AUTSX2) [MIM:300495]. AUTSX2 is a per- vasive developmental disorder (PDD), prototypically characterized by impairments in reciprocal social interaction and communication, Fig. 1. Residues 36-310 in 2wqzA colored by their relative importance. (See restricted and stereotyped patterns of interests and activities, and the Appendix, Fig.14, for the coloring scheme.) presence of developmental abnormalities by 3 years of age. Disease: Defects in NLGN4X may be the cause of susceptibility to X-linked Asperger syndrome 2 (ASPGX2) [MIM:300497]. ASPGX2 is considered to be a form of childhood autism. Similarity: Belongs to the type-B carboxylesterase/lipase 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 2wqzA For the chain 2wqzA, the alignment 2wqzA.msf (attached) with 819 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 311-598 in 2wqzA colored by their relative importance. (See duplicate sequences were removed. It can be found in the attachment Appendix, Fig.14, for the coloring scheme.) to this report, under the name of 2wqzA.msf. Its statistics, from the alistat program are the following: 2.3 Residue ranking in 2wqzA Format: MSF The 2wqzA sequence is shown in Figs. 1–2, with each residue colo- Number of sequences: 819 red according to its estimated importance. The full listing of residues Total number of residues: 350362 in 2wqzA can be found in the file called 2wqzA.ranks sorted in the Smallest: 61 attachment. Largest: 545 Average length: 427.8 2.4 Top ranking residues in 2wqzA and their position on Alignment length: 545 the structure Average identity: 31% In the following we consider residues ranking among top 25% of Most related pair: 99% residues in the protein . Figure 3 shows residues in 2wqzA colored Most unrelated pair: 0% by their importance: bright red and yellow indicate more conser- Most distant seq: 36% ved/important 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- gnment. 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the The alignment consists of 27% eukaryotic ( 12% vertebrata, top 25% of all residues, this time colored according to clusters they 11% arthropoda), and 2% prokaryotic sequences. (Descriptions of belong to. The clusters in Fig.4 are composed of the residues listed some sequences were not readily available.) The file containing the in Table 1. sequence descriptions can be found in the attachment, under the name 2wqzA.descr.

2 Table 1. continued cluster size member color residues red 131 57,73,74,75,76,77,78,79,81 82,84,86,87,88,90,91,103,110 112,144,145,146,147,148,149 150,151,152,154,167,168,169 170,171,172,173,174,175,176 177,180,182,187,190,191,194 196,197,198,199,200,202,203 204,205,206,208,209,210,211 212,213,220,221,223,224,225 226,227,230,231,233,234,237 238,241,242,243,244,245,248 249,250,251,252,253,254,255 256,259,260,263,266,267,269 271,272,273,275,276,277,279 280,281,306,317,318,342,345 366,370,372,375,376,446,449 452,468,470,472,489,492,495 497,498,516,527,530,531,534 536 Fig. 3. Residues in 2wqzA, colored by their relative importance. Clockwise: blue 2 53,97 front, back, top and bottom views. Table 1. Clusters of top ranking residues in 2wqzA.

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 2wqzD.Table 2 lists the top 25% of residues at the interface with 2wqzD. 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˚ ) 266 S S(75) 0.12 10/10 2.91 M(3) A(5)C .(7) T(3)VLG NIP(1)R HFYQ 534 G G(81) 0.13 1/1 4.80 S(3) .(11)AE QRDFTLP Fig. 4. Residues in 2wqzA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for YNMK the coloring scheme). Clockwise: front, back, top and bottom views. The 267 H P(73) 0.19 24/17 3.21 corresponding Pymol script is attached. H(3) K(1) .(7)S Table 1. E(3)G cluster size member M(1) color residues continued in next column continued in next column

3 Table 2. continued susbtantially larger than) other functional sites and interfaces reco- res type subst’s cvg noc/ dist gnizable in PDB entry 2wqz. It is shown in Fig. 6. The right panel (%) bb (A˚ ) shows (in blue) the rest of the larger cluster this surface belongs to. D(1) A(1)RL T(1)QVN Y

Table 2. The top 25% of residues in 2wqzA at the interface with 2wqzD. (Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type 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 of closest apporach to the ligand. )

Table 3. Fig. 6. A possible active surface on the chain 2wqzA. The larger cluster it res type disruptive belongs to is shown in blue. mutations 266 S (R)(K)(H)(Q) 534 G (R)(K)(E)(H) The residues belonging to this surface ”patch” are listed in Table 267 H (E)(T)(D)(Q) 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 4.6). Table 3. List of disruptive mutations for the top 25% of residues in Table 4. 2wqzA, that are at the interface with 2wqzD. res type substitutions(%) cvg antn 87 R R(92).(6)KWA 0.00 147 L L(94)I.(5) 0.00 77 P P(88)R(4).(6)GA 0.01 144 E E(92).(5)A(1)LD 0.01 PGS 79 A A(88)G(4).(6)SV 0.02 E 75 G G(89).(6)A(1)EK 0.03 STQRN 91 P P(85)A(4)LT(2)S 0.03 .(5)VG 97 W W(86).(5)F(2) 0.03 A(1)L(1)SRHDYGP T 241 F F(92).(7)YVA 0.03 88 F F(77)W(11).(5) 0.04 L(3)Y(1)P 145 D D(89)A.(5)GN(4) 0.04 KESHT 233 W W(91).(7)F(1)M 0.04 174 G G(89).(7)PWVEST 0.05 NLY 175 G G(84)E(6).(7)NA 0.05 TDQR 237 N N(86).(7)YH(4)I 0.06 Fig. 5. Residues in 2wqzA, at the interface with 2wqzD, colored by their rela- TEF tive importance. 2wqzD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2wqzA.) 242 G G(86).(7)HN(4)S 0.06 RKCAD 254 G S(78)G(11).(6)I 0.06 Figure 5 shows residues in 2wqzA colored by their importance, at the continued in next column interface with 2wqzD. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2wqzA surface, away from (or

4 Table 4. continued Table 4. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn D(1)ELFRAN 211 L L(70)F(15).(6) 0.13 53 G G(87).(10)ARESI 0.07 M(3)VSTAYIPQ LVKT 245 P P(79).(7)S(1) 0.13 112 Q Q(84)H(2).(5)GT 0.07 A(2)N(1)EK(2) YS(3)KAP(1)FLRD R(2)LQTVHG EIV 534 G G(81)S(3).(11)A 0.13 231 L L(79).(7)I(8) 0.07 EQRDFTLPYNMK M(4)FV 86 R KR(12)L(64)W 0.14 154 P P(81).(10)RT(2) 0.08 N(3).(8)MF(1) K(2)SAQGN H(1)Y(1)AG(2)DC 180 G G(83)N(4).(7)A 0.08 QSTV S(2)EKDCHF 263 L L(39)H(40).(7) 0.14 238 V I(80)A(6).(7) 0.08 Q(7)M(3)IANFVS V(4)L(1)CS 103 T V(3)A(66)T(6) 0.15 375 E E(81)N.(8)D(8)K 0.08 .(6)C(7)PS(1) PWGQAS G(4)DELFRYM 220 G G(80)DA(6).(7) 0.09 244 D D(68)N(22).(7)T 0.15 T(1)S(1)P(1)NEQ RAEKS L 171 Y W(61)F(14)Y(15) 0.16 281 G G(84).(7)A(3)P 0.09 .(7)IHVSTCNL S(1)V(1)NCLREFH 151 I I(34)V(56)L(3) 0.17 T .(5)M 173 H H(65)Y(19)F(5) 0.10 472 F F(74)Y(6).(8)N 0.17 .(7)P(2)ADC M(2)L(4)IDHWP 202 N N(67)Q(20)H.(7) 0.10 208 L L(54)W(3)F(30) 0.18 GS(1)EA(1)D .(7)M(2)IAEPH 272 L L(81).(7)AY(3) 0.10 225 L W(10)K(17)L(38) 0.18 V(1)KHM(2)FI(1) R(3)M(9)F(5) GTP .(6)T(1)Y(1)SE 273 F I(4)F(78)Y(2) 0.10 Q(1)H(1)AVI .(6)V(5)L(1)AS 492 E E(59).(9)D(28)N 0.18 489 H H(84).(9)SR(1)V 0.10 IGAKYQHT M(1)QKGDNIAL 148 Y Y(64)F(9)T(7) 0.19 81 P P(77)A(9).(6)Q 0.11 N(3).(5)S(2) K(2)TS(1)V R(1)V(1)K(2) 84 G G(75)V.(8)D(4) 0.11 H(1)CAWIQ E(1)RA(1)S(1)I 172 I I(71)V(8)F(6) 0.19 H(1)N(1)F(1) L(6).(7)STY K(1)QWC 267 H P(73)H(3)K(1) 0.19 152 Y W(28)Y(41)F(16) 0.12 .(7)SE(3)GM(1) H.(5)DME(1)V(3) D(1)A(1)RLT(1)Q T(1)AISL VNY 255 A VA(78)IS(3).(6) 0.12 271 G G(62)N(9)HP(3) 0.19 T(7)G(2)LD .(7)D(7)S(1)E 266 S S(75)M(3)A(5)C 0.12 R(1)AK(1)L(1) .(7)T(3)VLGNI Q(1)TV P(1)RHFYQ 317 C C(66)Q(1)A(4)K 0.20 S-S 345 D G(5)E(14)D(66) 0.12 F(5)L(2).(8)W .(7)VSNHTPRYKAL D(1)R(2)S(1) FQ T(1)IEGVYH 167 P P(69)T(3).(10) 0.13 366 D D(26)P(43)A(1) 0.20 A(12)S(2)DHK .(8)N(5)Q(5)F continued in next column continued in next column

5 Table 4. continued Table 5. res type substitutions(%) cvg antn res type disruptive S(1)E(4)KHTRVIG mutations 213 T A(6)VT(38)L(21) 0.21 87 R (TD)(YE)(S)(CG) H(6)M(1)P(2) 147 L (Y)(R)(T)(H) .(7)S(5)F(4)R 77 P (Y)(R)(H)(TE) I(1)G(1)EDNQ 144 E (H)(R)(FW)(Y) 253 S E(53)Q(13)S(3)G 0.21 79 A (R)(K)(Y)(H) H(7)N(2).(6) 75 G (R)(FW)(H)(E) D(1)Y(1)WFVTA 91 P (R)(Y)(H)(K) P(1)L(1)M(1)IRC 97 W (K)(E)(Q)(D) 497 F F(73)W(3).(9) 0.21 241 F (K)(E)(Q)(D) L(4)AT(2)V(1)QD 88 F (K)(E)(Q)(T) ISM(1)HRYGK 145 D (R)(FW)(H)(Y) 275 K R(59)K(16)Q(6) 0.22 233 W (KE)(T)(D)(Q) N(1).(6)A(1) 174 G (R)(K)(E)(H) G(4)S(1)YHT 175 G (R)(FWH)(K)(E) 318 L L(71)EM(9)F(2) 0.22 237 N (Y)(T)(HR)(FW) .(7)YTV(1)AI(2) 242 G (E)(R)(K)(FW) SKPQR 254 G (R)(K)(E)(H) 376 G G(52)A(10)F(8)P 0.22 53 G (R)(E)(K)(H) .(9)C(2)Y(3)I 112 Q (Y)(H)(FW)(T) S(6)L(1)MHNVWT 231 L (YR)(T)(H)(KE) E(1)QRKD 154 P (Y)(R)(H)(T) 74 L L(47)K(14)F(3) 0.23 180 G (R)(K)(E)(FWH) .(6)Q(3)E(3) 238 V (R)(K)(Y)(E) R(7)M(4)Y(2) 375 E (H)(FW)(Y)(R) T(4)VSN(1)DIHA 220 G (R)(KH)(FW)(E) 452 P P(60)G(10)S(3) 0.23 281 G (R)(K)(E)(H) .(8)IT(1)D(2) 173 H (E)(Q)(K)(MD) H(2)A(2)RQN(3)V 202 N (Y)(FWH)(T)(R) LCEFW 272 L (R)(Y)(H)(T) 90 P R(6)A(19)P(36) 0.24 273 F (K)(E)(Q)(R) E(2)LK(11)N(6) 489 H (E)(T)(D)(Q) .(5)MH(3)S(2) 81 P (Y)(R)(H)(T) D(1)QITYVG 84 G (ER)(K)(H)(D) 250 I L(39)I(40)V(12) 0.24 152 Y (K)(Q)(R)(M) .(6)HMA 255 A (R)(K)(Y)(E) 449 W F(58)W(4)I(5) 0.24 266 S (R)(K)(H)(Q) .(8)Y(7)V(6)N 345 D (R)(H)(FW)(Y) H(1)T(2)ARM(1) 167 P (R)(Y)(H)(T) L(1)SPKDC 211 L (R)(Y)(H)(K) 468 Y W(6)Y(66)F(12) 0.24 245 P (Y)(R)(H)(T) .(8)H(3)PNV(1)S 534 G (R)(K)(E)(H) RTG 86 R (D)(T)(E)(Y) 229 Q A(33)L(26)Q(7)T 0.25 263 L (Y)(R)(T)(H) .(7)M(13)K(2) 103 T (R)(K)(H)(Q) E(4)SR(1)HVFIYC 244 D (R)(FW)(H)(Y) 348 V L(7)V(23)T(2) 0.25 171 Y (K)(Q)(R)(E) I(4)F(45).(7)EA 151 I (Y)(R)(H)(T) MHDRWNY(1)S(1)Q 472 F (K)(E)(T)(Q) PG 208 L (R)(Y)(T)(H) continued in next column Table 4. Residues forming surface ”patch” in 2wqzA.

6 Table 5. continued res type disruptive mutations 225 L (Y)(R)(T)(H) 492 E (FWH)(R)(Y)(VA) 148 Y (K)(E)(Q)(M) 172 I (R)(Y)(K)(H) 267 H (E)(T)(D)(Q) 271 G (R)(E)(K)(H) 317 C (R)(K)(E)(H) 366 D (R)(H)(FW)(Y) 213 T (R)(K)(H)(FW) Fig. 7. Residues 82-288 in 2wqzC colored by their relative importance. (See 253 S (R)(K)(H)(Q) Appendix, Fig.14, for the coloring scheme.) 497 F (E)(K)(D)(T) 275 K (Y)(FW)(T)(VA) 318 L (Y)(R)(H)(T) About: This Swiss-Prot entry is copyright. It is produced through a 376 G (R)(K)(E)(H) collaboration between the Swiss Institute of Bioinformatics and the 74 L (Y)(R)(H)(T) EMBL outstation - the European Bioinformatics Institute. There are 452 P (R)(Y)(H)(T) no restrictions on its use as long as its content is in no way modified 90 P (Y)(R)(H)(T) and this statement is not removed. 250 I (Y)(R)(T)(H) 3.2 Multiple sequence alignment for 2wqzC 449 W (E)(K)(D)(T) For the chain 2wqzC, the alignment 2wqzC.msf (attached) with 39 468 Y (K)(Q)(EM)(R) sequences was used. The alignment was downloaded from the HSSP 229 Q (Y)(H)(T)(FW) database, and fragments shorter than 75% of the query as well as 348 V (R)(Y)(K)(E) duplicate sequences were removed. It can be found in the attachment to this report, under the name of 2wqzC.msf. Its statistics, from the Table 5. Disruptive mutations for the surface patch in 2wqzA. alistat program are the following:

Format: MSF Number of sequences: 39 3 CHAIN 2WQZC Total number of residues: 6721 3.1 P58400 overview Smallest: 86 Largest: 177 From SwissProt, id P58400, 98% identical to 2wqzC: Average length: 172.3 Description: Neurexin-1-beta precursor (Neurexin I-beta). Alignment length: 177 Organism, scientific name: Homo sapiens (Human). Average identity: 60% Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Most related pair: 99% Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Most unrelated pair: 15% Catarrhini; Hominidae; Homo. Most distant seq: 30% Function: Neuronal cell surface protein that may be involved in cell recognition and cell adhesion by forming intracellular juncti- ons through binding to neuroligins. May play a role in formation Furthermore, 3% of residues show as conserved in this alignment. or maintenance of synaptic junctions. May mediate intracellular The alignment consists of 33% eukaryotic ( 28% vertebrata, 5% signaling. arthropoda) sequences. (Descriptions of some sequences were not Subunit: The cytoplasmic C-terminal region binds to CASK. Iso- readily available.) The file containing the sequence descriptions can forms Beta 4b bind neuroligins NLGN1, NLGN2 and NLGN3, be found in the attachment, under the name 2wqzC.descr. alpha- dystroglycan and alpha-latrotoxin (By similarity). Subcellular location: Type I membrane protein (Potential). 3.3 Residue ranking in 2wqzC Alternative products: The 2wqzC sequence is shown in Fig. 7, with each residue colored Event=Alternative promoter; Comment=A number of isoforms according to its estimated importance. The full listing of residues are produced by use of alternative promoters. The alpha (AC in 2wqzC can be found in the file called 2wqzC.ranks sorted in the Q9ULB1) and beta isoforms (shown here) differ in their N- attachment. terminus; Event=Alternative splicing; Named isoforms=1; Com- ment=Experimental confirmation may be lacking for some isoforms; 3.4 Top ranking residues in 2wqzC and their position on Name=1; IsoId=P58400-1; Sequence=Displayed; the structure Ptm: Highly O-glycosylated and minor N-glycosylated (By simila- In the following we consider residues ranking among top 25% of rity). residues in the protein . Figure 8 shows residues in 2wqzC colored Similarity: Belongs to the neurexin family. by their importance: bright red and yellow indicate more conser- Similarity: Contains 1 laminin G-like domain. ved/important residues (see Appendix for the coloring scheme). A

7 Pymol script for producing this figure can be found in the attachment.

Fig. 9. Residues in 2wqzC, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Fig. 8. Residues in 2wqzC, colored by their relative importance. Clockwise: front, back, top and bottom views. Table 7. res type subst’s cvg noc/ dist 3.4.1 Clustering of residues at 25% coverage. Fig. 9 shows the (%) bb (A˚ ) top 25% of all residues, this time colored according to clusters they 235 T T(97) 0.12 13/3 3.04 belong to. The clusters in Fig.9 are composed of the residues listed .(2) in Table 6. 108 T T(92) 0.19 3/3 4.91 M(5) Table 6. .(2) cluster size member 234 L S(23) 0.24 24/2 3.09 color residues L(74) red 31 108,111,116,117,131,137,152 .(2) 153,155,158,170,171,173,174 232 R H(23) 0.25 6/0 3.32 176,177,180,182,184,185,187 R(74) 190,234,235,237,240,259,260 .(2) 261,263,266 blue 9 87,89,126,246,247,256,272 Table 7. The top 25% of residues in 2wqzC at the interface with 2wqzB. 286,287 (Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type Table 6. Clusters of top ranking residues in 2wqzC. 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 of closest apporach to the ligand. ) 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. Table 8. Interface with 2wqzB.Table 7 lists the top 25% of residues at res type disruptive the interface with 2wqzB. The following table (Table 8) suggests mutations possible disruptive replacements for these residues (see Section 4.6). 235 T (KR)(FQMWH)(NLPI)(E) 108 T (R)(KH)(FW)(Q) 234 L (R)(Y)(H)(T) continued in next column

8 Table 8. continued type in the bracket; noc/bb: number of contacts with the ligand, with the num- res type disruptive ber of contacts realized through backbone atoms given in the bracket; dist: mutations distance of closest apporach to the ligand. ) 232 R (TD)(SEVCLAPIG)(YM)(FNW)

Table 8. List of disruptive mutations for the top 25% of residues in Table 10. 2wqzC, that are at the interface with 2wqzB. res type disruptive mutations 155 G (E)(D)(FKMW)(YQLPHIR) 237 F (KE)(TQD)(SNCG)(R) 137 D (FWHR)(Y)(VCAG)(T) 234 L (R)(Y)(H)(T)

Table 10. List of disruptive mutations for the top 25% of residues in 2wqzC, that are at the interface with calcium ion.

Fig. 10. Residues in 2wqzC, at the interface with 2wqzB, colored by their relative importance. 2wqzB is shown in backbone representation (See Appen- dix for the coloring scheme for the protein chain 2wqzC.)

Figure 10 shows residues in 2wqzC colored by their importance, at the interface with 2wqzB. Calcium ion binding site. Table 9 lists the top 25% of residues at the interface with 2wqzCCA1289 (calcium ion). The following Fig. 11. Residues in 2wqzC, at the interface with calcium ion, colored by table (Table 10) suggests possible disruptive replacements for these their relative importance. The ligand (calcium ion) is colored green. Atoms residues (see Section 4.6). further than 30A˚ away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring Table 9. scheme for the protein chain 2wqzC.) res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 155 G G(97) 0.12 2/2 3.70 site Figure 11 shows residues in 2wqzC colored by their importance, at R(2) the interface with 2wqzCCA1289. 237 F F(97) 0.12 2/2 4.74 3.4.3 Possible novel functional surfaces at 25% coverage. One .(2) group of residues is conserved on the 2wqzC surface, away from (or 137 D D(97) 0.14 4/0 2.43 site susbtantially larger than) other functional sites and interfaces reco- Q(2) gnizable in PDB entry 2wqz. It is shown in Fig. 12. The residues 234 L S(23) 0.24 3/0 4.05 belonging to this surface ”patch” are listed in Table 11, while Table L(74) 12 suggests possible disruptive replacements for these residues (see .(2) Section 4.6).

Table 9. The top 25% of residues in 2wqzC at the interface with calcium ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each

9 Fig. 13. Another possible active surface on the chain 2wqzC. The larger cluster it belongs to is shown in blue.

Table 13. res type substitutions(%) cvg antn 153 N N(100) 0.03 173 Y Y(100) 0.03 176 V V(100) 0.03 184 N N(100) 0.03 155 G G(97)R(2) 0.12 site Fig. 12. A possible active surface on the chain 2wqzC. 158 D D(97)F(2) 0.12 170 D D(97)A(2) 0.12 Table 11. 174 H H(97)D(2) 0.12 180 R R(97)K(2) 0.12 res type substitutions(%) cvg 190 D D(97).(2) 0.12 272 A A(97).(2) 0.12 235 T T(97).(2) 0.12 287 L L(97).(2) 0.12 260 G G(97).(2) 0.12 256 G G(94)V(2).(2) 0.17 137 D D(97)Q(2) 0.14 site 87 Y Y(92).(7) 0.21 185 A A(97)S(2) 0.14 89 F F(92).(7) 0.21 131 S S(94)G(2)R(2) 0.15 286 R Q(25)R(71).(2) 0.23 116 G G(94)N(2).(2) 0.16 266 L L(94)A(2).(2) 0.17 Table 11. Residues forming surface ”patch” in 2wqzC. 261 L L(92)F(5).(2) 0.18 108 T T(92)M(5).(2) 0.19 152 F Y(25)F(74) 0.20 Table 12. 171 G N(25)G(74) 0.20 res type disruptive 177 R R(94)L(2)K(2) 0.23 mutations 263 Y V(25)Y(71).(2) 0.23 272 A (KYER)(QHD)(N)(FTMW) 234 L S(23)L(74).(2) 0.24 287 L (YR)(TH)(SCG)(KE) 186 T T(94)S(5) 0.25 256 G (KER)(HD)(Q)(FMW) 232 R H(23)R(74).(2) 0.25 87 Y (K)(QM)(NVLAPI)(ER) 89 F (KE)(TQD)(SNCG)(R) 286 R (T)(D)(YVCAG)(S) Table 13. Residues forming surface ”patch” in 2wqzC.

Table 12. Disruptive mutations for the surface patch in 2wqzC. Table 14. res type disruptive Another group of surface residues is shown in Fig.13. The right panel mutations shows (in blue) the rest of the larger cluster this surface belongs to. 153 N (Y)(FTWH)(SEVCARG)(MD) The residues belonging to this surface ”patch” are listed in Table 13, 173 Y (K)(QM)(NEVLAPIR)(D) while Table 14 suggests possible disruptive replacements for these 176 V (KYER)(QHD)(N)(FTMW) residues (see Section 4.6). 184 N (Y)(FTWH)(SEVCARG)(MD) 155 G (E)(D)(FKMW)(YQLPHIR) 158 D (R)(K)(CHG)(FTYVQAW) continued in next column

10 2 Table 14. continued at least 10A˚ , which is roughly the area needed for one water mole- res type disruptive cule to come in the contact with the residue. Furthermore, we require mutations that these residues form a “cluster” of residues which have neighbor 170 D (R)(H)(FKYW)(QCG) within 5A˚ from any of their heavy atoms. 174 H (TEQM)(KVCAG)(SNLPDI)(R) Note, however, that, if our picture of protein evolution is correct, 180 R (T)(YD)(SVCAG)(FELWPI) the neighboring residues which are not surface accessible might be 190 D (R)(FWH)(VCAG)(KY) equally important in maintaining the interaction specificity - they 235 T (KR)(FQMWH)(NLPI)(E) should not be automatically dropped from consideration when choo- 260 G (KER)(FQMWHD)(NLPI)(Y) sing the set for mutagenesis. (Especially if they form a cluster with 137 D (FWHR)(Y)(VCAG)(T) the surface residues.) 185 A (KR)(YE)(QH)(D) 131 S (K)(FMWR)(H)(EQ) 4.4 Number of contacts 116 G (R)(E)(K)(FWH) Another column worth noting is denoted “noc/bb”; it tells the num- 266 L (Y)(R)(H)(T) ber of contacts heavy atoms of the residue in question make across 261 L (R)(Y)(T)(H) the interface, as well as how many of them are realized through the 108 T (R)(KH)(FW)(Q) backbone atoms (if all or most contacts are through the backbone, 152 F (K)(E)(Q)(D) mutation presumably won’t have strong impact). Two heavy atoms 171 G (ER)(FKWH)(YMD)(Q) are considered to be “in contact” if their centers are closer than 5A˚ . 177 R (T)(Y)(D)(S) 4.5 Annotation 263 Y (K)(Q)(M)(E) 234 L (R)(Y)(H)(T) If the residue annotation is available (either from the pdb file or 186 T (KR)(FQMWH)(NELPI)(D) from other sources), another column, with the header “annotation” 232 R (TD)(SEVCLAPIG)(YM)(FNW) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 14. Disruptive mutations for the surface patch in 2wqzC. 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

11 5.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension COVERAGE (CE) of the optimal path . Protein Engineering 11(9) 739-747. 5.3.3 DSSP In this work a residue is considered solvent accessi- V ble if the DSSP program finds it exposed to water by at least 10A˚ 2, 100% 50% 30% 5% which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, http://www.cmbi.kun.nl/gv/dssp/descrip.html. V

RELATIVE IMPORTANCE 5.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de Fig. 14. Coloring scheme used to color residues by their relative importance. 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/ 2. their type 5.3.5 LaTex The text for this report was processed using LAT X; • rho ET score - the smaller this value, the lesser variability of E Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) • cvg coverage - percentage of the residues on the structure which 5.3.6 Muscle When making alignments “from scratch”, report have this rho or smaller maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 5.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 5.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. 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. 14. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 5.3 Credits visit: 5.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 5.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type

12 of service for comparative analysis of proteins.” Bioinformatics • 2wqzA.ranks sorted - full listing of residues and their ranking 22:1656-7. for 2wqzA • 2wqzA.2wqzD.if.pml - Pymol script for Figure 5 5.6 About report maker • 2wqzA.cbcvg - used by other 2wqzA – related pymol scripts report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 2wqzC.complex.pdb - coordinates of 2wqzC with all of its king visualization program was written by Ivica Res.ˇ report maker interacting partners is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. • 2wqzC.etvx - ET viewer input file for 2wqzC • 2wqzC.cluster report.summary - Cluster report summary for 5.7 Attachments 2wqzC The following files should accompany this report: • 2wqzC.ranks - Ranks file in sequence order for 2wqzC • 2wqzC.clusters - Cluster descriptions for 2wqzC • 2wqzA.complex.pdb - coordinates of 2wqzA with all of its • interacting partners 2wqzC.msf - the multiple sequence alignment used for the chain 2wqzC • 2wqzA.etvx - ET viewer input file for 2wqzA • 2wqzC.descr - description of sequences used in 2wqzC msf • 2wqzA.cluster report.summary - Cluster report summary for • 2wqzA 2wqzC.ranks sorted - full listing of residues and their ranking for 2wqzC • 2wqzA.ranks - Ranks file in sequence order for 2wqzA • 2wqzC.2wqzB.if.pml - Pymol script for Figure 10 • 2wqzA.clusters - Cluster descriptions for 2wqzA • 2wqzC.cbcvg - used by other 2wqzC – related pymol scripts • 2wqzA.msf - the multiple sequence alignment used for the chain • 2wqzA 2wqzC.2wqzCCA1289.if.pml - Pymol script for Figure 11 • 2wqzA.descr - description of sequences used in 2wqzA msf

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