Pages 1–6 2czy Evolutionary trace report by report maker July 31, 2009

4.3.1 Alistat 5 4.3.2 CE 6 4.3.3 DSSP 6 4.3.4 HSSP 6 4.3.5 LaTex 6 4.3.6 Muscle 6 4.3.7 Pymol 6 4.4 Note about ET Viewer 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 6

1 INTRODUCTION From the original Data Bank entry (PDB id 2czy): Title: Solution structure of the nrsf/rest-msin3b pah1 complex Compound: Mol id: 1; molecule: paired amphipathic helix protein sin3b; chain: a; fragment: pah1 domain (residues 31-107); synonym: sin3b, transcriptional sin3b, histone deacetylase complex subunit sin3b; engineered: yes; mol id: 2; molecule: transcription factor rest (version 3); chain: b; fragment: sin3 interaction domain (residues 43-57); synonym: nrsf/rest; engineered: yes CONTENTS Organism, scientific name: Mus Musculus; 2czy contains a single unique chain 2czyA (77 residues long). 1 Introduction 1 Chain 2czyB is too short (15 residues) to permit statistically signi- ficant analysis, and was treated as a peptide ligand. This is an 2 Chain 2czyA 1 NMR-determined structure – in this report the first model in the file 2.1 Q62141 overview 1 was used. 2.2 Multiple sequence alignment for 2czyA 1 2.3 Residue ranking in 2czyA 2 2 CHAIN 2CZYA 2.4 Top ranking residues in 2czyA and their position on 2.1 Q62141 overview the structure 2 2.4.1 Clustering of residues at 25% coverage. 2 From SwissProt, id Q62141, 100% identical to 2czyA: 2.4.2 Overlap with known functional surfaces at Description: Paired amphipathic helix protein Sin3b (Transcriptio- 25% coverage. 3 nal corepressor Sin3b) (Histone deacetylase complex subunit Sin3b). 2.4.3 Possible novel functional surfaces at 25% Organism, scientific name: Mus musculus (Mouse). coverage. 4 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Verte- brata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; 3 Notes on using trace results 4 Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus. 3.1 Coverage 4 Function: Acts as a transcriptional repressor. Interacts with MXI1 3.2 Known substitutions 4 to repress MYC responsive and antagonize MYC oncoge- 3.3 Surface 5 nic activities. Interacts with MAD-MAX heterodimers by binding 3.4 Number of contacts 5 to MAD. The heterodimer then represses transcription by tethering 3.5 Annotation 5 SIN3B to DNA. Also forms a complex with FOXK1 which represses 3.6 Mutation suggestions 5 transcription. Subunit: Interacts with HCFC1 (By similarity). Interacts with 4 Appendix 5 FOXK1/MNF, MXI1, MAD, NCOR1 and SAP30. Interaction with 4.1 File formats 5 SDS3 enhances the interaction with HDAC1 to form a complex. 4.2 Color schemes used 5 Subcellular location: Nuclear. 4.3 Credits 5 Alternative products:

1 Lichtarge lab 2006 2.4 Top ranking residues in 2czyA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 2czyA colored by their importance: bright red and yellow indicate more conser- Fig. 1. Residues 31-107 in 2czyA colored by their relative importance. (See ved/important residues (see Appendix for the coloring scheme). A Appendix, Fig.6, for the coloring scheme.) Pymol script for producing this figure can be found in the attachment.

Event=Alternative splicing; Named isoforms=4; Name=4; IsoId=Q62141-4; Sequence=Displayed; Note=No experimental con- firmation available; Name=1; IsoId=Q62141-1; Sequence=VSP 014187; Name=2; IsoId=Q62141-2; Sequence=VSP 008225, VSP 008226, VSP 014187; Name=3; IsoId=Q62141-3; Sequence=VSP 008227, VSP 008228, VSP 014187; Note=No experimental confir- mation available; Similarity: Contains 3 PAH (paired amphipathic helix) repeats. 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 2czyA For the chain 2czyA, the alignment 2czyA.msf (attached) with 107 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 2czyA.msf. Its statistics, from the alistat program are the following: Fig. 2. Residues in 2czyA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 107 Total number of residues: 7747 Smallest: 63 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Largest: 77 top 25% of all residues, this time colored according to clusters they Average length: 72.4 belong to. The clusters in Fig.3 are composed of the residues listed Alignment length: 77 in Table 1. Average identity: 49% Most related pair: 99% Table 1. Most unrelated pair: 9% cluster size member Most distant seq: 32% color residues red 19 37,40,44,45,48,55,58,62,66 75,79,82,83,89,92,93,96,97 Furthermore, <1% of residues show as conserved in this ali- 98 gnment. The alignment consists of 42% eukaryotic ( 6% vertebrata, 1% Table 1. Clusters of top ranking residues in 2czyA. arthropoda, 11% fungi, 19% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 2.4.2 Overlap with known functional surfaces at 25% coverage. 2czyA.descr. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. 2.3 Residue ranking in 2czyA Interface with the peptide 2czyB. Table 2 lists the top 25% of residues at the interface with 2czyB. The following table (Table The 2czyA sequence is shown in Fig. 1, with each residue colored 3) suggests possible disruptive replacements for these residues (see according to its estimated importance. The full listing of residues Section 3.6). in 2czyA can be found in the file called 2czyA.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) L(1) R(1) S(1) 66 K K(79) 0.22 300/7 2.07 S(1) Q(5) R(7)A G(1)YNC 40 Y Y(85) 0.25 14/0 4.08 F(11) .(1)A

Table 2. The top 25% of residues in 2czyA at the interface with 2czyB. (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. )

Fig. 3. Residues in 2czyA, colored according to the cluster they belong to: Table 3. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The res type disruptive corresponding Pymol script is attached. mutations 97 L (R)(Y)(H)(K) 93 F (KE)(T)(QDR)(SCG) Table 2. 37 A (R)(K)(E)(Q) res type subst’s cvg noc/ dist 75 V (R)(K)(E)(Y) (%) bb (A˚ ) 96 F (K)(E)(TQ)(DR) 97 L L(94) 0.04 32/2 3.17 58 F (KE)(T)(DR)(Q) V(2)T 62 M (Y)(T)(HR)(SCG) I(1) 98 P (Y)(R)(H)(T) 93 F F(96) 0.05 80/0 2.00 66 K (Y)(FW)(T)(VAHD) L(3) 40 Y (K)(Q)(EM)(NR) 37 A A(94)PC 0.08 113/18 2.80 GY.(1) Table 3. List of disruptive mutations for the top 25% of residues in 75 V V(89) 0.12 72/1 2.29 2czyA, that are at the interface with 2czyB. C(1) A(3) Figure 4 shows residues in 2czyA colored by their importance, at the L(1)YMG interface with 2czyB. 96 F F(93) 0.13 152/20 2.58 L(1) 2.4.3 Possible novel functional surfaces at 25% coverage. One I(1) group of residues is conserved on the 2czyA surface, away from (or Y(1)W susbtantially larger than) other functional sites and interfaces reco- 58 F F(93) 0.14 20/0 2.98 gnizable in PDB entry 2czy. It is shown in Fig. 5. The residues M(1) belonging to this surface ”patch” are listed in Table 4, while Table L(1)VIW 5 suggests possible disruptive replacements for these residues (see 62 M M(83) 0.17 189/22 1.98 Section 3.6). L(14)IF Table 4. V res type substitutions(%) cvg 98 P P(92) 0.18 16/0 4.21 89 L L(98)P(1) 0.01 .(1) 55 Y Y(97).F(1) 0.03 continued in next column 97 L L(94)V(2)TI(1) 0.04 93 F F(96)L(3) 0.05 continued in next column

3 Table 4. continued res type substitutions(%) cvg L(1)YMG 96 F F(93)L(1)I(1) 0.13 Y(1)W 58 F F(93)M(1)L(1)VI 0.14 W 62 M M(83)L(14)IFV 0.17 98 P P(92).(1)L(1) 0.18 R(1)S(1) 66 K K(79)S(1)Q(5) 0.22 R(7)AG(1)YNC 92 G G(84)A(1)D(2) 0.23 R(2)E(6)VT 40 Y Y(85)F(11).(1)A 0.25

Table 4. Residues forming surface ”patch” in 2czyA.

Table 5. res type disruptive mutations Fig. 4. Residues in 2czyA, at the interface with 2czyB, colored by their rela- 89 L (YR)(TH)(SKECG)(FQWD) tive importance. 2czyB is shown in backbone representation (See Appendix 55 Y (K)(Q)(M)(E) for the coloring scheme for the protein chain 2czyA.) 97 L (R)(Y)(H)(K) 93 F (KE)(T)(QDR)(SCG) 82 L (Y)(R)(TH)(E) 37 A (R)(K)(E)(Q) 45 K (Y)(FW)(T)(VA) 48 F (K)(E)(Q)(R) 75 V (R)(K)(E)(Y) 96 F (K)(E)(TQ)(DR) 58 F (KE)(T)(DR)(Q) 62 M (Y)(T)(HR)(SCG) 98 P (Y)(R)(H)(T) 66 K (Y)(FW)(T)(VAHD) 92 G (R)(K)(H)(E) 40 Y (K)(Q)(EM)(NR)

Table 5. Disruptive mutations for the surface patch in 2czyA.

3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to some small top percentage of residues [100% is all of the residues Fig. 5. A possible active surface on the chain 2czyA. in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or Table 4. continued to some nearby percentage), sorted by the strength of the presumed res type substitutions(%) cvg evolutionary pressure. (I.e., the smaller the coverage, the stronger the 82 L L(93)CMI(3)R 0.07 pressure on the residue.) Starting from the top of that list, mutating a 37 A A(94)PCGY.(1) 0.08 couple of residues should affect the protein somehow, with the exact 45 K K(90)R(5)SE(1)T 0.09 effects to be determined experimentally. 48 F F(88)L(5)CSY(3) 0.10 3.2 Known substitutions 75 V V(89)C(1)A(3) 0.12 One of the table columns is “substitutions” - other amino acid types continued in next column seen at the same position in the alignment. These amino acid types

4 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 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- sely, when looking for substitutions which will not affect the protein, COVERAGE one may try replacing, R with K, or (perhaps more surprisingly), with

V. The percentage of times the substitution appears in the alignment V is given in the immediately following bracket. No percentage is given 100% 50% 30% 5% in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up to 100%.

3.3 Surface V To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by RELATIVE IMPORTANCE 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor Fig. 6. Coloring scheme used to color residues by their relative importance. within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be choose, however, the straightforward alanine mutations, especially in equally important in maintaining the interaction specificity - they the beginning stages of their investigation. should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4 APPENDIX 3.4 Number of contacts 4.1 File formats Another column worth noting is denoted “noc/bb”; it tells the num- Files with extension “ranks sorted” are the actual trace results. The ber of contacts heavy atoms of the residue in question make across fields in the table in this file: the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • alignment# number of the position in the alignment mutation presumably won’t have strong impact). Two heavy atoms • residue# residue number in the PDB file ˚ are considered to be “in contact” if their centers are closer than 5A. • type amino acid type • 3.5 Annotation rank rank of the position according to older version of ET • variability If the residue annotation is available (either from the pdb file or has two subfields: from other sources), another column, with the header “annotation” 1. number of different amino acids appearing in in this column appears. Annotations carried over from PDB are the following: site of the alignment (indicating existence of related site record in PDB ), S-S (disulfide 2. their type bond forming residue), hb (hydrogen bond forming residue, jb (james • rho ET score - the smaller this value, the lesser variability of bond forming residue), and sb (for salt bridge forming residue). this position across the branches of the tree (and, presumably, the greater the importance for the protein) 3.6 Mutation suggestions • cvg coverage - percentage of the residues on the structure which Mutation suggestions are completely heuristic and based on comple- have this rho or smaller mentarity with the substitutions found in the alignment. Note that • gaps percentage of gaps in this column they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following properties: small [AV GSTC], medium [LPNQDEMIK], large 4.2 Color schemes used [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- The following color scheme is used in figures with residues colored tively [KHR], or negatively [DE] charged, aromatic [WFYH], by cluster size: black is a single-residue cluster; clusters composed of long aliphatic chain [EKRQM], OH-group possession [SDETY ], more than one residue colored according to this hierarchy (ordered and NH2 group possession [NQRK]. The suggestions are listed by descending size): red, blue, yellow, green, purple, azure, tur- according to how different they appear to be from the original amino quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, acid, and they are grouped in round brackets if they appear equally bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, disruptive. From left to right, each bracketed group of amino acid DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, types resembles more strongly the original (i.e. is, presumably, less tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive) These suggestions are tentative - they might prove disrup- The colors used to distinguish the residues by the estimated tive to the fold rather than to the interaction. Many researcher will evolutionary pressure they experience can be seen in Fig. 6.

5 4.3 Credits http://pymol.sourceforge.net/. (Note for Windows 4.3.1 Alistat alistat reads a multiple sequence alignment from the users: the attached package needs to be unzipped for Pymol to read file and shows a number of simple statistics about it. These stati- the scripts and launch the viewer.) stics include the format, the number of sequences, the total number 4.4 Note about ET Viewer of residues, the average and range of the sequence lengths, and the Dan Morgan from the Lichtarge lab has developed a visualization alignment length (e.g. including gap characters). Also shown are tool specifically for viewing trace results. If you are interested, please some percent identities. A percent pairwise alignment identity is defi- visit: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two http://mammoth.bcm.tmc.edu/traceview/ sequences. The ”average percent identity”, ”most related pair”, and The viewer is self-unpacking and self-installing. Input files to be used ”most unrelated pair” of the alignment are the average, maximum, with ETV (extension .etvx) can be found in the attachment to the and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant main report. seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N 4.5 Citing this work numbers (hence, the most outlying sequence). alistat is copyrighted The method used to rank residues and make predictions in this report by HHMI/Washington University School of Medicine, 1992-2001, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and freely distributed under the GNU General Public License. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 4.3.2 CE To map ligand binding sites from different by Importance” J. Mol. Bio. 336: 1265-82. For the original version source structures, report maker uses the CE program: of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tionary Trace Method Defines Binding Surfaces Common to Protein ”Protein structure alignment by incremental combinatorial extension Families” J. Mol. Bio. 257: 342-358. (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.3 DSSP In this work a residue is considered solvent accessi- of service for comparative analysis of .” Bioinformatics 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , 22:1656-7. 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. 4.6 About report maker Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version report maker was written in 2006 by Ivana Mihalek. The 1D ran- by [email protected] November 18,2002, king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, http://www.cmbi.kun.nl/gv/dssp/descrip.html. Houston. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than 4.7 Attachments 75% of the query are taken out, however); R. Schneider, A. de The following files should accompany this report: Daruvar, and C. Sander. ”The HSSP database of protein structure- • 2czyA.complex.pdb - coordinates of 2czyA with all of its sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. interacting partners http://swift.cmbi.kun.nl/swift/hssp/ • 2czyA.etvx - ET viewer input file for 2czyA • 4.3.5 LaTex The text for this report was processed using LATEX; 2czyA.cluster report.summary - Cluster report summary for Leslie Lamport, “LaTeX: A Document Preparation System Addison- 2czyA Wesley,” Reading, Mass. (1986). • 2czyA.ranks - Ranks file in sequence order for 2czyA 4.3.6 Muscle When making alignments “from scratch”, report • 2czyA.clusters - Cluster descriptions for 2czyA maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2czyA.msf - the multiple sequence alignment used for the chain ”MUSCLE: multiple sequence alignment with high accuracy and 2czyA high throughput.” Nucleic Acids Research 32(5), 1792-97. • 2czyA.descr - description of sequences used in 2czyA msf http://www.drive5.com/muscle/ • 2czyA.ranks sorted - full listing of residues and their ranking for 4.3.7 Pymol The figures in this report were produced using 2czyA Pymol. The scripts can be found in the attachment. Pymol • 2czyA.2czyB.if.pml - Pymol script for Figure 4 is an open-source application copyrighted by DeLano Scien- • 2czyA.cbcvg - used by other 2czyA – related pymol scripts tific LLC (2005). For more information about Pymol see

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