Pages 1–7 3b76 Evolutionary trace report by report maker April 17, 2009

4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 7 4.4 Note about ET Viewer 7 4.5 Citing this work 7 4.6 About report maker 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3b76): Title: Crystal structure of the third pdz domain of human ligand- of- numb protein-x (lnx1) in complex with the c-terminal peptide from the coxsackievirus and adenovirus receptor Compound: Mol id: 1; molecule: e3 ubiquitin-protein ligase lnx; chain: a, b; fragment: third pdz domain: residues 504-594; synonym: numb-binding protein 1, ligand of numb-protein x 1; ec: 6.3.2.-; engineered: yes CONTENTS Organism, scientific name: Homo Sapiens; 3b76 contains a single unique chain 3b76B (102 residues long) and 1 Introduction 1 its homologue 3b76A.

2 Chain 3b76B 1 2 CHAIN 3B76B 2.1 Q8TBB1 overview 1 2.1 Q8TBB1 overview 2.2 Multiple sequence alignment for 3b76B 1 2.3 Residue ranking in 3b76B 2 From SwissProt, id Q8TBB1, 100% identical to 3b76B: 2.4 Top ranking residues in 3b76B and their position on Description: Ubiquitin ligase LNX (EC 6.3.2.-) (Numb-binding the structure 2 protein 1) (Ligand of Numb-protein X 1). 2.4.1 Clustering of residues at 25% coverage. 2 Organism, scientific name: Homo sapiens (Human). 2.4.2 Overlap with known functional surfaces at Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 25% coverage. 2 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4.3 Possible novel functional surfaces at 25% Catarrhini; Hominidae; Homo. coverage. 4 Function: E3 Ubiquitin ligase protein that mediates ubiquitination and subsequent proteasomal degradation of NUMB. E3 ubiquitin 3 Notes on using trace results 5 ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme 3.1 Coverage 5 in the form of a thioester and then directly transfers the ubiquitin to 3.2 Known substitutions 6 targeted substrates. Mediates ubiquitination of isoform p66 and iso- 3.3 Surface 6 form p72 of NUMB, but not that of isoform p71 or isoform p65 (By 3.4 Number of contacts 6 similarity). 3.5 Annotation 6 Pathway: Ubiquitin conjugation; third step. 3.6 Mutation suggestions 6 Subunit: Interacts with the phosphotyrosine interaction domain of all isoforms of NUMB (By similarity). Interacts with the Coxsackie- 4 Appendix 6 virus and adenovirus receptor CXADR. Interacts with endogenous 4.1 File formats 6 retrovirus K protein Np9. 4.2 Color schemes used 6 Subcellular location: Cytoplasmic (By similarity). 4.3 Credits 7 Alternative products:

1 Lichtarge lab 2006 Event=Alternative splicing; Named isoforms=2; Name=1; IsoId=Q8TBB1-1; Sequence=Displayed; Name=2; IsoId=Q8TBB1- 2; Sequence=VSP 005733; Tissue specificity: Expressed in heart, placenta, kidney, pancreas and brain. Domain: The NPXY motif is required for the interaction with the PID domain of NUMB. It is however not sufficient. Domain: The PDZ 1 domain participates in the interaction with the PID domain of NUMB, and participates in the isoform-specific Fig. 1. Residues 401-502 in 3b76B colored by their relative importance. (See ubiquitination of NUMB. Appendix, Fig.7, for the coloring scheme.) Similarity: Contains 4 PDZ (DHR) domains. Similarity: Contains 1 RING-type zinc finger. About: This Swiss-Prot entry is copyright. It is produced through a Pymol script for producing this figure can be found in the attachment. 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 3b76B For the chain 3b76B, the alignment 3b76B.msf (attached) with 382 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 3b76B.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 382 Total number of residues: 32970 Smallest: 40 Largest: 102 Average length: 86.3 Alignment length: 102 Average identity: 31% Most related pair: 99% Most unrelated pair: 7% Most distant seq: 32%

Fig. 2. Residues in 3b76B, colored by their relative importance. Clockwise: Furthermore, <1% of residues show as conserved in this ali- front, back, top and bottom views. gnment. The alignment consists of 49% eukaryotic ( 39% vertebrata, 5% arthropoda) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the be found in the attachment, under the name 3b76B.descr. top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed 2.3 Residue ranking in 3b76B in Table 1. The 3b76B sequence is shown in Fig. 1, with each residue colored Table 1. according to its estimated importance. The full listing of residues cluster size member in 3b76B can be found in the file called 3b76B.ranks sorted in the color residues attachment. red 26 422,423,425,428,429,438,439 441,444,448,449,450,454,456 2.4 Top ranking residues in 3b76B and their position on 459,460,462,463,465,466,467 the structure 470,475,478,479,482 In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 3b76B colored Table 1. Clusters of top ranking residues in 3b76B. by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) E(1) A(1) .(5) L(2)CR T(1)HDK VYM 462 L I(50) 0.18 9/9 2.57 L(41) V(8). 470 L L(51) 0.19 12/6 3.81 I(2) V(21) F(15)C M(4) T(1)R.A 463 L I(12) 0.21 8/6 3.77 T(1) L(69) V(12) Fig. 3. Residues in 3b76B, colored according to the cluster they belong to: K(2)MPS 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. Table 2. The top 25% of residues in 3b76B at the interface with 1,2- ethanediol.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of 2.4.2 Overlap with known functional surfaces at 25% coverage. each type in the bracket; noc/bb: number of contacts with the ligand, with The name of the ligand is composed of the source PDB identifier the number of contacts realized through backbone atoms given in the bracket; and the heteroatom name used in that file. dist: distance of closest apporach to the ligand. ) 1,2-ethanediol binding site. Table 2 lists the top 25% of residues at the interface with 3b76BEDO601 (1,2-ethanediol). The following table (Table 3) suggests possible disruptive replacements for these Table 3. residues (see Section 3.6). res type disruptive mutations Table 2. 429 G (R)(E)(K)(H) res type subst’s cvg noc/ dist 439 I (R)(Y)(H)(E) ˚ (%) bb (A) 438 P (R)(Y)(H)(T) 429 G Y(2)N 0.12 1/1 4.55 462 L (YR)(H)(T)(KE) A(4) 470 L (Y)(R)(H)(E) G(68) 463 L (Y)(R)(H)(T) Q(6) R(6) Table 3. List of disruptive mutations for the top 25% of residues in .(3) 3b76B, that are at the interface with 1,2-ethanediol. D(2) E(2)VSC IPHFK Figure 4 shows residues in 3b76B colored by their importance, at the 439 I I(76)AC 0.14 18/11 3.17 interface with 3b76BEDO601. V(12) Interface with 3b76A.Table 4 lists the top 25% of residues at the Q(1) interface with 3b76A. The following table (Table 5) suggests possible .(1)G disruptive replacements for these residues (see Section 3.6). L(6)FTP Table 4. 438 P G(46)F 0.17 12/8 3.52 res type subst’s cvg noc/ dist P(29)Q (%) bb (A˚ ) S(8) 423 G G(97) 0.01 6/6 2.92 continued in next column .(1)CSQ continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) R(10) P(2) L(2)ECD GIS.M 479 V V(72) 0.16 16/2 3.84 S(1) A(9)CT Q(3) M(1) K(1) L(2) I(4)R.N 438 P G(46)F 0.17 15/1 3.18 P(29)Q S(8) E(1) A(1) .(5) L(2)CR Fig. 4. Residues in 3b76B, at the interface with 1,2-ethanediol, colored by T(1)HDK their relative importance. The ligand (1,2-ethanediol) is colored green. Atoms VYM further than 30A˚ away from the geometric center of the ligand, as well as on 463 L I(12) 0.21 5/0 3.14 the line of sight to the ligand were removed. (See Appendix for the coloring T(1) scheme for the protein chain 3b76B.) L(69) V(12) Table 4. continued K(2)MPS res type subst’s cvg noc/ dist . (%) bb (A˚ ) 482 L L(80) 0.09 15/5 3.96 Table 4. The top 25% of residues in 3b76B at the interface with 3b76A. F(6) (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 in the bracket; noc/bb: number of contacts with the ligand, with the number of I(9)EV. contacts realized through backbone atoms given in the bracket; dist: distance 422 L L(77) 0.11 24/11 2.75 of closest apporach to the ligand. ) M(2) F(10) .(2)T Table 5. I(2) res type disruptive Y(1) mutations V(1) 423 G (R)(KE)(FWH)(MD) W(1)HPC 482 L (R)(Y)(TH)(CG) 428 G G(81) 0.13 13/13 2.84 422 L (R)(Y)(K)(TE) A(3)K 428 G (R)(K)(E)(H) S(3) 475 R (T)(D)(Y)(E) Y(2) 479 V (Y)(R)(E)(H) T(1)R 438 P (R)(Y)(H)(T) V(1)M 463 L (Y)(R)(H)(T) .(1)ELC DI 475 R N(9) 0.15 23/0 2.96 Table 5. List of disruptive mutations for the top 25% of residues in Y(1) 3b76B, that are at the interface with 3b76A. Q(11) F(1) Figure 5 shows residues in 3b76B colored by their importance, at the H(57) interface with 3b76A. continued in next column 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 3b76B surface, away from (or

4 Table 6. continued res type substitutions(%) cvg . 448 G S(16)G(77)PL(1) 0.06 TMQ(1)FA(1)K 454 G G(92)V(1)KS(2)A 0.07 NEDQRL 478 A A(79)C(1)V(11)K 0.08 RL(2)T(1)IS(1)P E. 482 L L(80)F(6)M(2) 0.09 I(9)EV. 422 L L(77)M(2)F(10) 0.11 .(2)TI(2)Y(1) V(1)W(1)HPC 429 G Y(2)NA(4)G(68) 0.12 Q(6)R(6).(3) D(2)E(2)VSCIPHF K 428 G G(81)A(3)KS(3) 0.13 Y(2)T(1)RV(1)M .(1)ELCDI Fig. 5. Residues in 3b76B, at the interface with 3b76A, colored by their rela- 439 I I(76)ACV(12) 0.14 tive importance. 3b76A is shown in backbone representation (See Appendix Q(1).(1)GL(6)FT for the coloring scheme for the protein chain 3b76B.) P 475 R N(9)Y(1)Q(11) 0.15 F(1)H(57)R(10) susbtantially larger than) other functional sites and interfaces reco- P(2)L(2)ECDGIS. gnizable in PDB entry 3b76. It is shown in Fig. 6. The right panel M shows (in blue) the rest of the larger cluster this surface belongs to. 479 V V(72)S(1)A(9)CT 0.16 Q(3)M(1)K(1) L(2)I(4)R.N 438 P G(46)FP(29)Q 0.17 S(8)E(1)A(1) .(5)L(2)CRT(1)H DKVYM 462 L I(50)L(41)V(8). 0.18 470 L L(51)I(2)V(21) 0.19 F(15)CM(4)T(1)R .A 456 I I(32)L(57)V(6) 0.20 M(2)F. 463 L I(12)T(1)L(69) 0.21 Fig. 6. A possible active surface on the chain 3b76B. The larger cluster it V(12)K(2)MPS. belongs to is shown in blue. 444 V V(48)MTI(44)H 0.23 L(3)CS(1)A. 449 V A(49)L(4)P(14) 0.24 The residues belonging to this surface ”patch” are listed in Table V(14)I(4)Q(1) 6, while Table 7 suggests possible disruptive replacements for these S(1)K(1)D(1) residues (see Section 3.6). M(1)C(2)ET Table 6. res type substitutions(%) cvg Table 6. Residues forming surface ”patch” in 3b76B. 423 G G(97).(1)CSQ 0.01 460 D D(95)M(1)EIQ.LG 0.02 NY 459 G N(8)G(87)DKYEHC 0.03 continued in next column

5 2 Table 7. 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 423 G (R)(KE)(FWH)(MD) within 5A˚ from any of their heavy atoms. 460 D (R)(H)(FW)(Y) Note, however, that, if our picture of protein evolution is correct, 459 G (R)(K)(E)(FW) the neighboring residues which are not surface accessible might be 448 G (R)(E)(K)(H) equally important in maintaining the interaction specificity - they 454 G (R)(H)(FEW)(K) should not be automatically dropped from consideration when choo- 478 A (Y)(R)(K)(E) sing the set for mutagenesis. (Especially if they form a cluster with 482 L (R)(Y)(TH)(CG) the surface residues.) 422 L (R)(Y)(K)(TE) 429 G (R)(E)(K)(H) 3.4 Number of contacts 428 G (R)(K)(E)(H) Another column worth noting is denoted “noc/bb”; it tells the num- 439 I (R)(Y)(H)(E) ber of contacts heavy atoms of the residue in question make across 475 R (T)(D)(Y)(E) the interface, as well as how many of them are realized through the 479 V (Y)(R)(E)(H) backbone atoms (if all or most contacts are through the backbone, 438 P (R)(Y)(H)(T) mutation presumably won’t have strong impact). Two heavy atoms 462 L (YR)(H)(T)(KE) are considered to be “in contact” if their centers are closer than 5A˚ . 470 L (Y)(R)(H)(E) 3.5 Annotation 456 I (YR)(T)(H)(KE) 463 L (Y)(R)(H)(T) If the residue annotation is available (either from the pdb file or 444 V (R)(K)(E)(Y) from other sources), another column, with the header “annotation” 449 V (Y)(R)(H)(K) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 7. Disruptive mutations for the surface patch in 3b76B. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- 3.1 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 3.2 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 4 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- 4.1 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 3.3 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

6 4.3.2 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. 4.3.3 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 4.3.4 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. 7. 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 4.3.5 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 4.3.6 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. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 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 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 7. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 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 4.5 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

7 of service for comparative analysis of proteins.” Bioinformatics • 3b76B.cluster report.summary - Cluster report summary for 22:1656-7. 3b76B • 3b76B.ranks - Ranks file in sequence order for 3b76B 4.6 About report maker • 3b76B.clusters - Cluster descriptions for 3b76B report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 3b76B.msf - the multiple sequence alignment used for the chain king visualization program was written by Ivica Res.ˇ report maker 3b76B is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. • 3b76B.descr - description of sequences used in 3b76B msf • 3b76B.ranks sorted - full listing of residues and their ranking 4.7 Attachments for 3b76B The following files should accompany this report: • 3b76B.3b76BEDO601.if.pml - Pymol script for Figure 4 • 3b76B.cbcvg - used by other 3b76B – related pymol scripts • 3b76B.complex.pdb - coordinates of 3b76B with all of its • interacting partners 3b76B.3b76A.if.pml - Pymol script for Figure 5 • 3b76B.etvx - ET viewer input file for 3b76B

8