Pages 1–5 1bbi Evolutionary trace report by report maker September 26, 2008

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

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1bbi): Title: Three-dimensional structure of soybean trypsin(slash) chy- motrypsin bowman-birk inhibitor in solution Compound: Mol id: 1; molecule: trypsin/chymotrypsin bowman- birk inhibitor; chain: a; engineered: yes Organism, scientific name: Max CONTENTS 1bbi contains a single unique chain 1bbiA (71 residues long). This 1 Introduction 1 is an NMR-determined structure – in this report the first model in the file was used. 2 Chain 1bbiA 1 2.1 Q84LF5 overview 1 2.2 Multiple sequence alignment for 1bbiA 1 2.3 Residue ranking in 1bbiA 1 2.4 Top ranking residues in 1bbiA and their position on 2 CHAIN 1BBIA the structure 1 2.1 Q84LF5 overview 2.4.1 Clustering of residues at 25% coverage. 1 2.4.2 Possible novel functional surfaces at 25% From SwissProt, id Q84LF5, 100% identical to 1bbiA: coverage. 2 Description: Bowman-Birk protease inhibitor. Organism, scientific name: Glycine microphylla. 3 Notes on using trace results 3 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 3.1 Coverage 3 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3.2 Known substitutions 3 eudicotyledons; ; eurosids I; ; ; Papilionoi- 3.3 Surface 3 deae; ; Glycine. 3.4 Number of contacts 3 3.5 Annotation 3 3.6 Mutation suggestions 3 2.2 Multiple sequence alignment for 1bbiA 4 Appendix 4 For the chain 1bbiA, the alignment 1bbiA.msf (attached) with 27 4.1 File formats 4 sequences was used. The alignment was assembled through combi- 4.2 Color schemes used 4 nation of BLAST searching on the UniProt database and alignment 4.3 Credits 4 using Muscle program. It can be found in the attachment to this 4.3.1 Alistat 4 report, under the name of 1bbiA.msf. Its statistics, from the alistat 4.3.2 CE 4 program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-71 in 1bbiA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.)

Format: MSF Number of sequences: 27 Total number of residues: 1816 Smallest: 60 Largest: 71 Average length: 67.3 Alignment length: 71 Average identity: 63% Most related pair: 99% Most unrelated pair: 40% Most distant seq: 60%

Furthermore, 25% of residues show as conserved in this alignment. Fig. 2. Residues in 1bbiA, colored by their relative importance. Clockwise: The alignment consists of 96% eukaryotic ( 96% plantae) front, back, top and bottom views. sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 1bbiA.descr.

2.3 Residue ranking in 1bbiA The 1bbiA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1bbiA can be found in the file called 1bbiA.ranks sorted in the attachment.

2.4 Top ranking residues in 1bbiA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1bbiA colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the 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 in Table 1.

Table 1. cluster size member Fig. 3. Residues in 1bbiA, colored according to the cluster they belong to: color residues red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The red 7 8,9,12,14,22,58,62 corresponding Pymol script is attached. blue 3 36,39,51 yellow 3 17,19,20 green 2 44,46 purple 2 41,49 2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bbiA surface, away from (or Table 1. Clusters of top ranking residues in 1bbiA. susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1bbi. It is shown in Fig. 4. The residues belonging to this surface ”patch” are listed in Table 2, while Table

2 Table 3. continued res type disruptive mutations 17 S (KR)(FQMWH)(NYELPI)(D) 19 P (YR)(TH)(SKECG)(FQWD) 20 P (YR)(TH)(SKECG)(FQWD) 22 C (KER)(FQMWHD)(NYLPI)(SVA) 33 H (E)(TQMD)(SNKVCLAPIG)(YR) 36 C (KER)(FQMWHD)(NYLPI)(SVA) 39 C (KER)(FQMWHD)(NYLPI)(SVA) 41 C (KER)(FQMWHD)(NYLPI)(SVA) 49 C (KER)(FQMWHD)(NYLPI)(SVA) 51 C (KER)(FQMWHD)(NYLPI)(SVA) 58 C (KER)(FQMWHD)(NYLPI)(SVA) 62 C (KER)(FQMWHD)(NYLPI)(SVA)

Table 3. Disruptive mutations for the surface patch in 1bbiA.

3 NOTES ON USING TRACE RESULTS

Fig. 4. A possible active surface on the chain 1bbiA. 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 3 suggests possible disruptive replacements for these residues (see some small top percentage of residues [100% is all of the residues Section 3.6). 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 2. to some nearby percentage), sorted by the strength of the presumed res type substitutions(%) cvg antn evolutionary pressure. (I.e., the smaller the coverage, the stronger the 8 C C(100) 0.25 S-S pressure on the residue.) Starting from the top of that list, mutating a 9 C C(100) 0.25 S-S couple of residues should affect the protein somehow, with the exact 12 C C(100) 0.25 S-S effects to be determined experimentally. 14 C C(100) 0.25 S-S 17 S S(100) 0.25 site 3.2 Known substitutions 19 P P(100) 0.25 20 P P(100) 0.25 One of the table columns is “substitutions” - other amino acid types 22 C C(100) 0.25 S-S seen at the same position in the alignment. These amino acid types 33 H H(100) 0.25 may be interchangeable at that position in the protein, so if one wants 36 C C(100) 0.25 S-S to affect the protein by a point mutation, they should be avoided. For 39 C C(100) 0.25 S-S example if the substitutions are “RVK” and the original protein has 41 C C(100) 0.25 S-S an R at that position, it is advisable to try anything, but RVK. Conver- n 49 C C(100) 0.25 S-S sely, when looking for substitutions which will ot affect the protein, 51 C C(100) 0.25 S-S one may try replacing, R with K, or (perhaps more surprisingly), with 58 C C(100) 0.25 S-S V. The percentage of times the substitution appears in the alignment 62 C C(100) 0.25 S-S is given in the immediately following bracket. No percentage is given 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 Table 2. Residues forming surface ”patch” in 1bbiA. to 100%. 3.3 Surface Table 3. res type disruptive To detect candidates for novel functional interfaces, first we look for mutations residues that are solvent accessible (according to DSSP program) by A˚ 2 8 C (KER)(FQMWHD)(NYLPI)(SVA) at least 10 , which is roughly the area needed for one water mole- 9 C (KER)(FQMWHD)(NYLPI)(SVA) cule to come in the contact with the residue. Furthermore, we require 12 C (KER)(FQMWHD)(NYLPI)(SVA) that these residues form a “cluster” of residues which have neighbor A˚ 14 C (KER)(FQMWHD)(NYLPI)(SVA) within 5 from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, continued in next column the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they

3 should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts COVERAGE Another column worth noting is denoted “noc/bb”; it tells the num-

ber of contacts heavy atoms of the residue in question make across V the interface, as well as how many of them are realized through the 100% 50% 30% 5% backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . 3.5 Annotation

If the residue annotation is available (either from the pdb file or V from other sources), another column, with the header “annotation” RELATIVE IMPORTANCE appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james Fig. 5. Coloring scheme used to color residues by their relative importance. bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions • gaps percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein The following color scheme is used in figures with residues colored with its ligand. The attempt is made to complement the following by cluster size: black is a single-residue cluster; clusters composed of properties: small [AV GST C], medium [LP NQDEMIK], large more than one residue colored according to this hierarchy (ordered [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- by descending size): red, blue, yellow, green, purple, azure, tur- tively [KHR], or negatively [DE] charged, aromatic [W F Y H], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, long aliphatic chain [EKRQM], OH-group possession [SDET Y ], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, and NH2 group possession [NQRK]. The suggestions are listed DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, according to how different they appear to be from the original amino tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. acid, and they are grouped in round brackets if they appear equally The colors used to distinguish the residues by the estimated disruptive. From left to right, each bracketed group of amino acid evolutionary pressure they experience can be seen in Fig. 5. types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 4 APPENDIX alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of Files with extension “ranks sorted” are the actual trace results. The exact identities and len1, len2 are the unaligned lengths of the two fields in the table in this file: sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • residue# residue number in the PDB file seq” is calculated by finding the maximum pairwise identity (best • type amino acid type relative) for all N sequences, then finding the minimum of these N • rank rank of the position according to older version of ET numbers (hence, the most outlying sequence). alistat is copyrighted • variability has two subfields: by HHMI/Washington University School of Medicine, 1992-2001, 1. number of different amino acids appearing in in this column and freely distributed under the GNU General Public License. of the alignment 4.3.2 CE To map ligand binding sites from different 2. their type source structures, report maker uses the CE program: http://cl.sdsc.edu/ • rho ET score - the smaller this value, the lesser variability of . Shindyalov IN, Bourne PE (1998) this position across the branches of the tree (and, presumably, ”Protein structure alignment by incremental combinatorial extension the greater the importance for the protein) (CE) of the optimal path . Protein Engineering 11(9) 739-747. • cvg coverage - percentage of the residues on the structure which 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 have this rho or smaller ble if the DSSP program finds it exposed to water by at least 10A˚ ,

4 which is roughly the area needed for one water molecule to come in The viewer is self-unpacking and self-installing. Input files to be used the contact with the residue. DSSP is copyrighted by W. Kabsch, C. with ETV (extension .etvx) can be found in the attachment to the Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version main report. by [email protected] November 18,2002, 4.5 Citing this work http://www.cmbi.kun.nl/gv/dssp/descrip.html. The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 4.3.4 HSSP Whenever available, report maker uses HSSP ali- Evolution-Entropy Hybrid Methods for Ranking of Protein Residues gnment as a starting point for the analysis (sequences shorter than by Importance” J. Mol. Bio. 336: 1265-82. For the original version 75% of the query are taken out, however); R. Schneider, A. de of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- Daruvar, and C. Sander. ”The HSSP database of protein structure- tionary Trace Method Defines Binding Surfaces Common to Protein sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. Families” J. Mol. Bio. 257: 342-358. report maker itself is described in Mihalek I., I. Res and O. http://swift.cmbi.kun.nl/swift/hssp/ Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics 4.3.5 LaTex A The text for this report was processed using LTEX; 22:1656-7. Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- 4.3.6 Muscle When making alignments “from scratch”, report king visualization program was written by Ivica Res.ˇ report maker maker uses Muscle alignment program: Edgar, Robert C. (2004), is copyrighted by Lichtarge Lab, Baylor College of Medicine, ”MUSCLE: multiple sequence alignment with high accuracy and Houston. high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.7 Attachments http://www.drive5.com/muscle/ The following files should accompany this report: • 1bbiA.complex.pdb - coordinates of 1bbiA with all of its inter- 4.3.7 Pymol The figures in this report were produced using acting partners Pymol. The scripts can be found in the attachment. Pymol • is an open-source application copyrighted by DeLano Scien- 1bbiA.etvx - ET viewer input file for 1bbiA tific LLC (2005). For more information about Pymol see • 1bbiA.cluster report.summary - Cluster report summary for http://pymol.sourceforge.net/. (Note for Windows 1bbiA users: the attached package needs to be unzipped for Pymol to read • 1bbiA.ranks - Ranks file in sequence order for 1bbiA the scripts and launch the viewer.) • 1bbiA.clusters - Cluster descriptions for 1bbiA 4.4 Note about ET Viewer • 1bbiA.msf - the multiple sequence alignment used for the chain Dan Morgan from the Lichtarge lab has developed a visualization 1bbiA tool specifically for viewing trace results. If you are interested, please • 1bbiA.descr - description of sequences used in 1bbiA msf visit: • 1bbiA.ranks sorted - full listing of residues and their ranking for 1bbiA http://mammoth.bcm.tmc.edu/traceview/

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