Pages 1–7 1hfo Evolutionary trace report by report maker July 28, 2010

4.3.1 Alistat 6 4.3.2 CE 6 4.3.3 DSSP 6 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 1hfo): Title: The structure of the macrophage migration inhibitory factor from spiralis. CONTENTS Compound: Mol id: 1; molecule: migration inhibitory factor; chain: a, b, c, d, e, f; engineered: yes 1 Introduction 1 Organism, scientific name: ; 1hfo contains a single unique chain 1hfoA (113 residues long) and 2 Chain 1hfoA 1 its homologues 1hfoF, 1hfoD, 1hfoC, 1hfoE, and 1hfoB. 2.1 Q9Y063 overview 1 2.2 Multiple sequence alignment for 1hfoA 1 2.3 Residue ranking in 1hfoA 1 2.4 Top ranking residues in 1hfoA and their position on the structure 1 2.4.1 Clustering of residues at 25% coverage. 1 2 CHAIN 1HFOA 2.4.2 Overlap with known functional surfaces at 2.1 Q9Y063 overview 25% coverage. 2 2.4.3 Possible novel functional surfaces at 25% From SwissProt, id Q9Y063, 98% identical to 1hfoA: coverage. 4 Description: Macrophage migration inhibitory factor like protein. Organism, scientific name: Trichinella spiralis (Trichina worm). 3 Notes on using trace results 5 : Eukaryota; Metazoa; Nematoda; ; Trichocepha- 3.1 Coverage 5 lida; Trichinellidae; Trichinella. 3.2 Known substitutions 5 3.3 Surface 5 3.4 Number of contacts 6 3.5 Annotation 6 2.2 Multiple sequence alignment for 1hfoA 3.6 Mutation suggestions 6 For the chain 1hfoA, the alignment 1hfoA.msf (attached) with 117 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 6 database, and fragments shorter than 75% of the query as well as 4.1 File formats 6 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 6 to this report, under the name of 1hfoA.msf. Its statistics, from the 4.3 Credits 6 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-113 in 1hfoA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Format: MSF Number of sequences: 117 Total number of residues: 13047 Smallest: 95 Largest: 113 Average length: 111.5 Alignment length: 113 Average identity: 36% Most related pair: 99% Most unrelated pair: 17% Most distant seq: 35% Fig. 2. Residues in 1hfoA, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 33% eukaryotic ( 14% vertebrata, <1% arthropoda, 3% plantae), and 3% prokaryotic sequences. (Des- criptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1hfoA.descr. 2.3 Residue ranking in 1hfoA The 1hfoA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1hfoA can be found in the file called 1hfoA.ranks sorted in the attachment. 2.4 Top ranking residues in 1hfoA 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 1hfoA 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 Fig. 3. Residues in 1hfoA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for in Table 1. the coloring scheme). Clockwise: front, back, top and bottom views. The Table 1. corresponding Pymol script is attached. cluster size member color residues red 15 1,3,23,27,30,32,33,36,37,39 Table 1. continued 63,64,65,112,113 cluster size member blue 7 7,8,49,51,55,93,95 color residues continued in next column yellow 4 72,76,79,98

Table 1. Clusters of top ranking residues in 1hfoA.

2 2.4.2 Overlap with known functional surfaces at 25% coverage. Table 3. continued The name of the ligand is composed of the source PDB identifier res type disruptive and the heteroatom name used in that file. mutations Interface with 1hfoC.Table 2 lists the top 25% of residues at the 107 G (KR)(E)(QH)(FMWD) interface with 1hfoC. The following table (Table 3) suggests possible 37 V (R)(Y)(KE)(H) disruptive replacements for these residues (see Section 3.6). 113 F (TKE)(D)(SQCRG)(N) 112 T (R)(K)(H)(Q) Table 2. 23 S (R)(K)(H)(Q) res type subst’s cvg noc/ dist antn 39 V (R)(K)(Y)(E) (%) bb (A˚ ) 1 P P(97) 0.04 10/9 3.77 site Table 3. List of disruptive mutations for the top 25% of residues in .(2) 1hfoA, that are at the interface with 1hfoC. 36 Y Y(84) 0.04 22/15 2.58 R(12)FC W 107 G G(78) 0.09 52/52 2.75 A(16)S .(4) 37 V V(64) 0.12 20/20 2.91 I(29) L(1) M(2)C 113 F F(77) 0.16 8/4 4.04 .(8) M(9) L(4) 112 T T(82) 0.17 51/20 2.98 .(5)I V(6) P(1) L(2) 23 S T(32) 0.23 7/0 3.55 S(49) C(7) A(1) E(3)GI H(1).P 39 V V(57) 0.25 44/38 2.78 Fig. 4. Residues in 1hfoA, at the interface with 1hfoC, colored by their rela- I(15) tive importance. 1hfoC is shown in backbone representation (See Appendix T(19) for the coloring scheme for the protein chain 1hfoA.) A(3) S(2) L(1) Figure 4 shows residues in 1hfoA colored by their importance, at the interface with 1hfoC. Interface with 1hfoB.Table 4 lists the top 25% of residues at the Table 2. The top 25% of residues in 1hfoA at the interface with 1hfoC. interface with 1hfoB. The following table (Table 5) suggests possible (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 disruptive replacements for these residues (see Section 3.6). in the bracket; noc/bb: number of contacts with the ligand, with the number of Table 4. contacts realized through backbone atoms given in the bracket; dist: distance res type subst’s cvg noc/ dist of closest apporach to the ligand. ) (%) bb (A˚ ) 93 R R(94)HY 0.06 2/2 4.39 Table 3. K(3) res type disruptive 72 N N(70) 0.08 23/7 3.11 mutations M(2) 1 P (YR)(TH)(SCG)(KE) T(16) 36 Y (K)(Q)(E)(M) .(6) continued in next column continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) K(2)A 51 G G(89) 0.13 5/5 4.03 D(2)T A(4) K(1)N 98 F F(87)L 0.18 41/17 2.84 Y(9)VS. 76 S S(78) 0.19 34/10 2.89 A(9) T(9)RIN 95 Y Y(80)P 0.20 80/6 2.78 L(3) F(5)M I(3) V(4)H 49 F F(78) 0.21 28/10 3.49 L(3) V(4) Y(3) H(4)R Fig. 5. Residues in 1hfoA, at the interface with 1hfoB, colored by their rela- W(3) tive importance. 1hfoB is shown in backbone representation (See Appendix M(1) for the coloring scheme for the protein chain 1hfoA.)

Table 4. The top 25% of residues in 1hfoA at the interface with 1hfoB. (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 5. res type disruptive mutations 93 R (TD)(EVA)(SCLPIG)(YM) 72 N (Y)(H)(FW)(T) 51 G (R)(FWH)(KE)(Y) 98 F (K)(E)(Q)(DR) 76 S (R)(K)(H)(FW) 95 Y (K)(QR)(E)(M) 49 F (E)(K)(TD)(Q)

Table 5. List of disruptive mutations for the top 25% of residues in 1hfoA, that are at the interface with 1hfoB. Fig. 6. A possible active surface on the chain 1hfoA. Figure 5 shows residues in 1hfoA colored by their importance, at the interface with 1hfoB.

2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1hfoA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1hfo. It is shown in Fig. 6. The residues belonging to this surface ”patch” are listed in Table 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 3.6).

4 Table 6. 9 suggests possible disruptive replacements for these residues (see res type substitutions(%) cvg Section 3.6). 93 R R(94)HYK(3) 0.06 55 P P(92)G(2)KL(2) 0.07 Table 8. D(1) res type substitutions(%) cvg antn 8 N N(76)S(21).(2) 0.11 32 K K(98)NT 0.01 51 G G(89)D(2)TA(4) 0.13 33 P P(97)S(2) 0.02 K(1)N 65 G G(96)NAD(1) 0.03 95 Y Y(80)PL(3)F(5)M 0.20 1 P P(97).(2) 0.04 site I(3)V(4)H 36 Y Y(84)R(12)FCW 0.04 49 F F(78)L(3)V(4) 0.21 72 N N(70)M(2)T(16) 0.08 Y(3)H(4)RW(3) .(6)K(2)A M(1) 107 G G(78)A(16)S.(4) 0.09 27 G A(84)S(6)VG(5)C 0.10 P Table 6. Residues forming surface ”patch” in 1hfoA. 37 V V(64)I(29)L(1) 0.12 M(2)C 64 I I(84)V(7)L(5)MT 0.15 Table 7. 113 F F(77).(8)M(9) 0.16 res type disruptive L(4) mutations 112 T T(82).(5)IV(6) 0.17 93 R (TD)(EVA)(SCLPIG)(YM) P(1)L(2) 55 P (Y)(R)(H)(T) 98 F F(87)LY(9)VS. 0.18 8 N (Y)(FWH)(TR)(EVCAG) 76 S S(78)A(9)T(9)RI 0.19 51 G (R)(FWH)(KE)(Y) N 95 Y (K)(QR)(E)(M) 79 L L(42)I(26)F(24) 0.20 49 F (E)(K)(TD)(Q) V(5) 30 L L(49)I(12)M(11) 0.22 Table 7. Disruptive mutations for the surface patch in 1hfoA. T(20)V(1)YC(1)F 23 S T(32)S(49)C(7) 0.23 A(1)E(3)GIH(1). Another group of surface residues is shown in Fig.7. The residues P 3 F F(35)L(23)I(23) 0.24 V(11)C(2).(2) 39 V V(57)I(15)T(19) 0.25 A(3)S(2)L(1)

Table 8. Residues forming surface ”patch” in 1hfoA.

Table 9. res type disruptive mutations 32 K (Y)(FW)(T)(VAH) 33 P (R)(Y)(H)(K) 65 G (R)(K)(EH)(FW) 1 P (YR)(TH)(SCG)(KE) 36 Y (K)(Q)(E)(M) 72 N (Y)(H)(FW)(T) 107 G (KR)(E)(QH)(FMWD) 27 G (R)(K)(E)(H) 37 V (R)(Y)(KE)(H) 64 I (R)(Y)(H)(TK) 113 F (TKE)(D)(SQCRG)(N) 112 T (R)(K)(H)(Q) 98 F (K)(E)(Q)(DR) Fig. 7. Another possible active surface on the chain 1hfoA. continued in next column belonging to this surface ”patch” are listed in Table 8, while Table

5 Table 9. continued the interface, as well as how many of them are realized through the res type disruptive backbone atoms (if all or most contacts are through the backbone, mutations mutation presumably won’t have strong impact). Two heavy atoms 76 S (R)(K)(H)(FW) are considered to be “in contact” if their centers are closer than 5A˚ . 79 L (R)(Y)(T)(KEH) 30 L (R)(Y)(KH)(E) 3.5 Annotation 23 S (R)(K)(H)(Q) If the residue annotation is available (either from the pdb file or 3 F (KE)(QR)(D)(T) from other sources), another column, with the header “annotation” 39 V (R)(K)(Y)(E) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 9. Disruptive mutations for the surface patch in 1hfoA. 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 Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino couple of residues should affect the protein somehow, with the exact acid, and they are grouped in round brackets if they appear equally effects to be determined experimentally. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 4 APPENDIX 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, one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file: 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 guide - due to rounding errors these percentages often do not add up • residue# residue number in the PDB file to 100%. • type amino acid type 3.3 Surface • rank rank of the position according to older version of ET • To detect candidates for novel functional interfaces, first we look for variability has two subfields: residues that are solvent accessible (according to DSSP program) by 1. number of different amino acids appearing in in this column 2 at least 10A˚ , which is roughly the area needed for one water mole- of the alignment cule to come in the contact with the residue. Furthermore, we require 2. their type that these residues form a “cluster” of residues which have neighbor • rho ET score - the smaller this value, the lesser variability of within 5A˚ from any of their heavy atoms. this position across the branches of the tree (and, presumably, Note, however, that, if our picture of protein evolution is correct, the greater the importance for the protein) the neighboring residues which are not surface accessible might be • cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with • gaps percentage of gaps in this column the surface residues.) 4.2 Color schemes used 3.4 Number of contacts The following color scheme is used in figures with residues colored Another column worth noting is denoted “noc/bb”; it tells the num- by cluster size: black is a single-residue cluster; clusters composed of ber of contacts heavy atoms of the residue in question make across more than one residue colored according to this hierarchy (ordered

6 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 Daruvar, and C. Sander. ”The HSSP database of protein structure- COVERAGE sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/ V A 100% 50% 30% 5% 4.3.5 LaTex The text for this report was processed using LTEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004),

V ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. RELATIVE IMPORTANCE http://www.drive5.com/muscle/

Fig. 8. Coloring scheme used to color residues by their relative importance. 4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol 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. 4.4 Note about ET Viewer The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 8. 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- 4.5 Citing this work ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report ˇ sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res, 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 of service for comparative analysis of proteins.” Bioinformatics 4.3.2 CE To map ligand binding sites from different 22:1656-7. source structures, report maker uses the CE program: 4.6 About report maker http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension report maker was written in 2006 by Ivana Mihalek. The 1D ran- (CE) of the optimal path . Protein Engineering 11(9) 739-747. king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.3 DSSP In this work a residue is considered solvent accessi- Houston. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in 4.7 Attachments the contact with the residue. DSSP is copyrighted by W. Kabsch, C. The following files should accompany this report: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version • 1hfoA.complex.pdb - coordinates of 1hfoA with all of its inter- by [email protected] November 18,2002, acting partners http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1hfoA.etvx - ET viewer input file for 1hfoA

7 • 1hfoA.cluster report.summary - Cluster report summary for • 1hfoA.ranks sorted - full listing of residues and their ranking for 1hfoA 1hfoA • 1hfoA.ranks - Ranks file in sequence order for 1hfoA • 1hfoA.1hfoC.if.pml - Pymol script for Figure 4 • 1hfoA.clusters - Cluster descriptions for 1hfoA • 1hfoA.cbcvg - used by other 1hfoA – related pymol scripts • 1hfoA.msf - the multiple sequence alignment used for the chain • 1hfoA.1hfoB.if.pml - Pymol script for Figure 5 1hfoA • 1hfoA.descr - description of sequences used in 1hfoA msf

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