Pages 1–5 3b7x Evolutionary trace report by report maker May 28, 2010

4.3.3 DSSP 4 4.3.4 HSSP 4 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5

1 INTRODUCTION From the original Data Bank entry (PDB id 3b7x): Title: Crystal structure of human fk506-binding protein 6 Compound: Mol id: 1; molecule: fk506-binding protein 6; chain: a; fragment: ppiase -type domain: residues 12-144; synonym: peptidyl-prolyl cis-trans , ppiase, rotamase, 36 kda fk506- binding protein, fkbp-36, immunophilin fkbp36; ec: 5.2.1.8; engi- neered: yes Organism, scientific name: Homo Sapiens; 3b7x contains a single unique chain 3b7xA (117 residues long).

CONTENTS 2 CHAIN 3B7XA 2.1 O75344 overview 1 Introduction 1 From SwissProt, id O75344, 93% identical to 3b7xA: 2 Chain 3b7xA 1 Description: FK506-binding protein 6 (EC 5.2.1.8) (Peptidyl-prolyl 2.1 O75344 overview 1 cis-trans isomerase) (PPIase) (Rotamase) (36 kDa FK506 binding 2.2 Multiple sequence alignment for 3b7xA 1 protein) (FKBP- 36) (Immunophilin FKBP36). 2.3 Residue ranking in 3b7xA 1 Organism, scientific name: Homo sapiens (Human). 2.4 Top ranking residues in 3b7xA and their position on Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; the structure 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4.1 Clustering of residues at 25% coverage. 2 Catarrhini; Hominidae; Homo. 2.4.2 Possible novel functional surfaces at 25% Function: PPIases accelerate the folding of . coverage. 2 Catalytic activity: Peptidylproline (omega=180) = peptidylproline (omega=0). 3 Notes on using trace results 3 Tissue specificity: Detected in all tissues examined, with higher 3.1 Coverage 3 expression in testis, heart, skeletal muscle, liver, and kidney. 3.2 Known substitutions 3 Disease: Haploinsufficiency of FKBP6 may be the cause of cer- 3.3 Surface 3 tain cardiovascular and musculo-skeletal abnormalities observed in 3.4 Number of contacts 3 Williams-Beuren syndrome (WBS) [MIM:194050]; a rare develop- 3.5 Annotation 4 mental disorder. It is a contiguous deletion syndrome involving 3.6 Mutation suggestions 4 from chromosome band 7q11.23. Similarity: Contains 1 PPIase FKBP-type domain. 4 Appendix 4 Similarity: Contains 3 TPR repeats. 4.1 File formats 4 About: This Swiss-Prot entry is copyright. It is produced through a 4.2 Color schemes used 4 collaboration between the Swiss Institute of Bioinformatics and the 4.3 Credits 4 EMBL outstation - the European Bioinformatics Institute. There are 4.3.1 Alistat 4 no restrictions on its use as long as its content is in no way modified 4.3.2 CE 4 and this statement is not removed.

1 Lichtarge lab 2006 Fig. 1. Residues 19-143 in 3b7xA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.)

2.2 Multiple sequence alignment for 3b7xA For the chain 3b7xA, the alignment 3b7xA.msf (attached) with 41 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 3b7xA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 41 Total number of residues: 4182 Fig. 2. Residues in 3b7xA, colored by their relative importance. Clockwise: front, back, top and bottom views. Smallest: 88 Largest: 117 Average length: 102.0 Alignment length: 117 Average identity: 46% Most related pair: 99% Most unrelated pair: 20% Most distant seq: 49%

Furthermore, 4% of residues show as conserved in this alignment. The alignment consists of 63% eukaryotic ( 41% vertebrata, 7% arthropoda, 7% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descrip- tions can be found in the attachment, under the name 3b7xA.descr. 2.3 Residue ranking in 3b7xA The 3b7xA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 3b7xA can be found in the file called 3b7xA.ranks sorted in the attachment. 2.4 Top ranking residues in 3b7xA and their position on the structure

In the following we consider residues ranking among top 25% of Fig. 3. Residues in 3b7xA, colored according to the cluster they belong to: residues in the protein . Figure 2 shows residues in 3b7xA colored red, followed by blue and yellow are the largest clusters (see Appendix for by their importance: bright red and yellow indicate more conser- the coloring scheme). Clockwise: front, back, top and bottom views. The ved/important residues (see Appendix for the coloring scheme). A corresponding Pymol script is attached. Pymol script for producing this figure can be found in the attachment.

Table 1. 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the cluster size member top 25% of all residues, this time colored according to clusters they color residues belong to. The clusters in Fig.3 are composed of the residues listed continued in next column in Table 1.

2 Table 1. continued Table 2. continued cluster size member res type substitutions(%) cvg color residues 35 D D(92)N(4).(2) 0.15 red 28 35,40,46,61,63,72,94,98,102 125 P P(90)S(2)N(2) 0.16 105,106,108,110,112,114,116 D(2)Q(2) 117,118,119,122,125,127,128 135 F F(90)W(2)Y(7) 0.17 129,132,135,136,138 138 E E(90)Q(2)H(2) 0.19 .(2)K(2) Table 1. Clusters of top ranking residues in 3b7xA. 119 G G(92)K(2)R(2) 0.20 H(2) 129 P P(70)G(4)S(21) 0.21 2.4.2 Possible novel functional surfaces at 25% coverage. One A(2) group of residues is conserved on the 3b7xA surface, away from (or 72 F F(85)Y(4).(7) 0.23 susbtantially larger than) other functional sites and interfaces reco- S(2) gnizable in PDB entry 3b7x. It is shown in Fig. 4. The right panel 136 E E(78)K(9)D(9) 0.25 shows (in blue) the rest of the larger cluster this surface belongs to. I(2)

Table 2. Residues forming surface ”patch” in 3b7xA.

Table 3. res type disruptive mutations 40 K (Y)(FTW)(SVCAG)(HD) 106 E (FWH)(YVCARG)(T)(SNKLPI) 122 G (KER)(FQMWHD)(NYLPI)(SVA) 128 P (Y)(T)(HR)(SCG) 127 I (YR)(H)(TKE)(SQCDG) 118 Y (K)(Q)(EM)(NR) Fig. 4. A possible active surface on the chain 3b7xA. The larger cluster it 46 G (KR)(E)(QH)(FMW) belongs to is shown in blue. 114 P (R)(Y)(H)(T) 105 G (K)(R)(E)(QM) 116 Y (K)(R)(Q)(E) The residues belonging to this surface ”patch” are listed in Table 117 A (R)(KYE)(H)(QD) 2, while Table 3 suggests possible disruptive replacements for these 61 Y (K)(Q)(M)(NER) residues (see Section 3.6). 132 T (R)(K)(H)(FW) Table 2. 35 D (R)(FWH)(YVCAG)(T) res type substitutions(%) cvg 125 P (Y)(R)(H)(T) 40 K K(100) 0.04 135 F (K)(E)(Q)(D) 106 E E(100) 0.04 138 E (FW)(YVAH)(CG)(T) 122 G G(100) 0.04 119 G (E)(D)(FMW)(KY) 128 P P(90)K(9) 0.05 129 P (R)(Y)(H)(K) 127 I I(97)V(2) 0.06 72 F (K)(E)(Q)(D) 118 Y Y(87)F(12) 0.07 136 E (FWH)(Y)(CRG)(VA) 46 G A(7)G(90)S(2) 0.08 114 P P(85)K(2)Y(9) 0.09 Table 3. Disruptive mutations for the surface patch in 3b7xA. S(2) 105 G G(90)Y(7)S(2) 0.10 116 Y Y(82)L(14)V(2) 0.11 117 A A(80)V(2)L(9) 0.12 3 NOTES ON USING TRACE RESULTS G(7) 3.1 Coverage 61 Y Y(92)S(2)F(2) 0.14 .(2) Trace results are commonly expressed in terms of coverage: the resi- 132 T T(80)D(9)E(2) 0.14 due is important if its “coverage” is small - that is if it belongs to A(2)V(4) some small top percentage of residues [100% is all of the residues continued in next column 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 to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the

3 pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3.2 Known substitutions COVERAGE One of the table columns is “substitutions” - other amino acid types

seen at the same position in the alignment. These amino acid types V may be interchangeable at that position in the protein, so if one wants 100% 50% 30% 5% 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, 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 RELATIVE IMPORTANCE 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%. Fig. 5. Coloring scheme used to color residues by their relative importance. 3.3 Surface To detect candidates for novel functional interfaces, first we look for according to how different they appear to be from the original amino residues that are solvent accessible (according to DSSP program) by acid, and they are grouped in round brackets if they appear equally 2 at least 10A˚ , which is roughly the area needed for one water mole- disruptive. From left to right, each bracketed group of amino acid cule to come in the contact with the residue. Furthermore, we require types resembles more strongly the original (i.e. is, presumably, less that these residues form a “cluster” of residues which have neighbor disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4.1 File formats Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • ber of contacts heavy atoms of the residue in question make across alignment# number of the position in the alignment the interface, as well as how many of them are realized through the • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 3.5 Annotation 1. number of different amino acids appearing in in this column If the residue annotation is available (either from the pdb file or of the alignment from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • gaps percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,

4 DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, 4.3.7 Pymol The figures in this report were produced using tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. Pymol. The scripts can be found in the attachment. Pymol The colors used to distinguish the residues by the estimated is an open-source application copyrighted by DeLano Scien- evolutionary pressure they experience can be seen in Fig. 5. tific LLC (2005). For more information about Pymol see http://pymol.sourceforge.net/. (Note for Windows 4.3 Credits users: the attached package needs to be unzipped for Pymol to read 4.3.1 Alistat alistat reads a multiple sequence alignment from the the scripts and launch the viewer.) file and shows a number of simple statistics about it. These stati- 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 http://mammoth.bcm.tmc.edu/traceview/ exact identities and len1, len2 are the unaligned lengths of the two 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 4.5 Citing this work relative) for all N sequences, then finding the minimum of these N 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 by Importance” J. Mol. Bio. 336: 1265-82. For the original version 4.3.2 CE To map ligand binding sites from different of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- source structures, report maker uses the CE program: tionary Trace Method Defines Binding Surfaces Common to Protein http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) Families” J. Mol. Bio. 257: 342-358. ”Protein structure alignment by incremental combinatorial extension report maker itself is described in Mihalek I., I. Res and O. (CE) of the optimal path . Protein Engineering 11(9) 739-747. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 22:1656-7. ble if the DSSP program finds it exposed to water by at least 10A˚ , which is roughly the area needed for one water molecule to come in 4.6 About report maker the contact with the residue. DSSP is copyrighted by W. Kabsch, C. report maker was written in 2006 by Ivana Mihalek. The 1D ran- Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version king visualization program was written by Ivica Res.ˇ report maker @cmbi.kun.nl by Elmar.Krieger November 18,2002, is copyrighted by Lichtarge Lab, Baylor College of Medicine, http://www.cmbi.kun.nl/gv/dssp/descrip.html. Houston. 4.7 Attachments 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than The following files should accompany this report: 75% of the query are taken out, however); R. Schneider, A. de • 3b7xA.complex.pdb - coordinates of 3b7xA with all of its Daruvar, and C. Sander. ”The HSSP database of protein structure- interacting partners sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 3b7xA.etvx - ET viewer input file for 3b7xA http://swift.cmbi.kun.nl/swift/hssp/ • 3b7xA.cluster report.summary - Cluster report summary for 3b7xA 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 3b7xA.ranks - Ranks file in sequence order for 3b7xA Wesley,” Reading, Mass. (1986). • 3b7xA.clusters - Cluster descriptions for 3b7xA • 4.3.6 Muscle When making alignments “from scratch”, report 3b7xA.msf - the multiple sequence alignment used for the chain maker uses Muscle alignment program: Edgar, Robert C. (2004), 3b7xA ”MUSCLE: multiple sequence alignment with high accuracy and • 3b7xA.descr - description of sequences used in 3b7xA msf high throughput.” Nucleic Acids Research 32(5), 1792-97. • 3b7xA.ranks sorted - full listing of residues and their ranking http://www.drive5.com/muscle/ for 3b7xA

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