SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES

SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES

Anna Grzechnik1, Dennis Carlton1, Heath Klock2 Mark W. Knuth2 and Scott A. Lesley1,2*

1 The Joint Center for Structural Genomics (JCSG), The Scripps Research Institute

2 JCSG, The Genomics Institute of the Novartis Research Institute *[email protected]

NIH Bottlenecks Meeting4/15/08

• Protein families come in all shapes and sizes

• Common traits which allow us to recognize them but individual characteristics can vary greatly

• Protein families are collections of related sequences.

• Draft 1 and 2 Pfam families are large with many potential targets.

With 100 or more potential family members, we have the pick of the litter

We want these targets.

Not these targets.

Target Selection Within Pfam Assignments

PG1132I - 2.00 A resolution

Practical Filters: genomic DNA, #met, #cys

8 targets with crystals to beamline

87 targets pursued from PF07726

http://ffas.burnham.org/XtalPred-cgi/xtal.pl

Just doing more targets is not enough.

Select 82 families from 400 Draft 1 and 2 targets which failed using multiple targets.

Find the best possible targets from all genomes and process through a full panel of salvage strategies.

Low Temperature Expression

Regular Temperature Expression Solubility (37C)1 2 3 4 5 6 7 8 9 10 11 12

A -0.023 -0.024 -0.021 0.113 -0.020 -0.021 0.367 0.586 -0.015 0.012 0.233 -0.032

B -0.012 0.322 -0.023 0.834 0.027 0.706 0.057 -0.024 0.043 0.474 -0.001 -0.018

C -0.006 0.335 -0.009 0.065 0.004 0.008 0.010 0.006 -0.032 0.003 0.244 -0.009

D -0.002 -0.004 0.013 -0.009 0.509 -0.025 -0.039 -0.012 -0.009 0.145 -0.009 -0.037

E 0.019 -0.002 0.041 0.128 0.622 0.335 0.029 0.322 0.163 0.241 -0.013 -0.005

F 0.154 0.357 -0.004 0.029 0.000 0.076 -0.014 0.002 -0.021 -0.012 0.014 0.023

G 1.023 0.103 0.006 -0.003 0.033 -0.036 0.049 0.002 0.033 0.037 0.053 0.436

H 0.280 0.021 0.030 0.002 0.056 0.019 0.006 0.000

Total: 24

Low Temperature Expression Solubility (25C)1 2 3 4 5 6 7 8 9 10 11 12

A 0.038 0.196 0.047 0.617 0.083 0.386 0.423 0.428 0.186 0.179 0.580 0.106

B 0.455 0.517 0.133 0.511 0.309 0.522 0.562 0.204 0.334 0.455 0.102 0.141

C 0.129 0.393 0.246 0.296 0.156 0.187 0.240 0.174 0.137 0.182 0.131 0.055

D 0.223 0.101 0.196 0.196 0.676 0.162 0.592 0.181 0.375 0.052 0.101 0.090

E 0.044 0.137 0.111 0.340 0.523 0.761 0.305 0.556 0.295 0.095 0.076 0.081

F 0.346 0.343 0.459 0.117 0.105 0.118 0.168 0.252 0.083 0.250 0.163 0.148

G 0.782 0.620 0.140 0.134 0.195 0.061 0.490 0.115 0.168 0.077 0.213 0.569

H 0.392 0.166 0.098 0.131 0.262 0.219 0.248 0.274

Total: 78

37C

25C

188 Targets from failed 82 families

Microscreen expression and purification with total yield solubility cutoff

Low Temperature Expression

Micro-ANSEC analysis. Highly parallel, quick (12 min) run times, minimal resolution for aggregation testing

Low Temperature Expression

Making Truncations

Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 7: 982-94.

PIPE cloning facilitates making truncations and point mutationsWhat truncations to make?

•nested N- and C-terminal•bioinformatic predictions•experimentally determined

1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG 150 151 ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211 ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267

Un

cleaved

Clea

ved

1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG 150 151 ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211 ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267

Overlapping fragment not pursued further.

31790.801

18035.201

9017.601

PT03787B-mth-267-18-182

Partial Proteolysis

PT03787B-mth-267-46-127

Fragment was reconstructed, re-expressedand is currently in crystal trials.

Deuterium Exchange Mass Spectrometry (DXMS)

DXMS identifies regions of disorder and flexibility by mapping the location of rapid hydrogen/deuterium exchange to peptides derived from targets of interest. The information can be used to design expression constructs with improved crystallization properties (Spraggon et al., 2004).

PFAM 6249: Ethanolamine utilization protein eutQ from Salmonella typhimurium LT2

FL-protein: poor crystallization Truncation: 1.9Å structure

High-Exchange Location WT Targets Salvage ConstructsClass Number % N I C To Beam Solved Solved Rate Solved Salvage rate Total Solved Overall Success

I 30 10% n/a n/a n/a 16 13 43.3% 0 0.0% 13 43.3%II 106 35% 51 85 55 43 16 15.1% 7 7.8% 23 21.7%III 86 28% 58 78 59 32 7 8.1% 4 5.1% 11 12.8%IV 77 25% 44 27 48 32 5 6.5% 12 16.7% 17 22.1%V 8 3% 8 8 8 1 0 0.0% 0 0.0% 0 0.0%

307 161 198 170 124 41 23 64 20.8%

I

II

III

IV

V

Surface Mutagenesis

Ligand ScreeningTarget: PG1132F

Target PG1132F binds Blue resin suggesting nucleotide binding. Target is screened against nucleotide ligand library by thermofluor. Binding to ADP and GDP is observed.

185 Targets

74 Non binding targets

Targets that do not bind to the resin are screened against a 300 ligand library

111 Targets bindAffi-Gel Blue

Method courtesy of Chang Yub-Kim

Methods of ligand screening used:•Thermofluor: fluorescent detection of melting temperature•DSC: detection of differences in protein heat capacity•Stargazer: light scattering detection of aggregation

Nucleotide-LikeLigand Library:ADP GDPADP Ribose GTPAMP NADATP NADPCDP NADPH CMP TryptophanCTPcAMP

Thermofluor Ligand Screen on PG1132F

0.00

10000.00

20000.00

30000.00

40000.00

50000.00

60000.00

70000.00

20 30 40 50 60 70 80 90 100

Temperature (C)

Flu

ore

scen

ce None

ADP

GDP

Target PS04248

RM

Target PS03963

No mountable crystals were available from native protein. After reductive

methylation, two conditions produced harvestable crystals which are currently in finescreening.

Structure of PS04248 was solved with Reductive Methylation

RM

Reductive Methylation

UCSD & BurnhamBioinformatics CoreJohn WooleyAdam GodzikLukasz JaroszewskiSri Krishna SubramanianAndrew MorseTamara AstakhovaLian DuanPiotr KozbialDana WeekesNatasha SefcovicPrasad BurraJosie AlaoenCindy Cook

GNF & TSRICrystallomics CoreScott LesleyMark KnuthHeath KlockDennis CarltonThomas ClaytonChristina TroutMarc DellerDaniel McMullanPolat Abdubek Julie FeuerhelmJoanna HaleJessica PaulsenThamara JanaratneHope JohnsonEdward NigoghossianLinda OkachSebastian SudekGlen SpraggonSanjay AgarwallaAnna GrzechnikRegina GorskiConnie ChenDustin Ernst

TSRINMR CoreKurt Wüthrich Reto Horst Maggie JohnsonAmaranth ChatterjeeMichael GeraltWojtek AugustyniakPedro SerranoBill PedriniWilliam Placzek

TSRI Administrative CoreIan WilsonMarc ElsligerGye Won HanDavid MarcianoHenry TienLisa van Veen

Stanford /SSRLStructure Determination CoreKeith HodgsonAshley DeaconMitchell Miller Herbert AxelrodHsiu-Ju (Jessica) ChiuKevin JinChristopher RifeQingping XuSilvya OommachenHenry van den BedemScott TalafuseRonald ReyesAbhinav KumarChristine Trame

The JCSG is supported by the NIH Protein Structure Initiative (PSI) Grant U54 GM074898 from NIGMS (www.nigms.nih.gov).