Structural Genomics and the Protein Folding Problem George N. Phillips, Jr. University of Wisconsin-Madison February 15, 2006

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference From DNA to biological function

Developing a gene model Glimmer (Gene Locator and Interpolated Markov ModelER) GlimmerHMM for eukaryotic genomes (more advanced) Genome sequencing Genome assembly Regulatory elements Identification of ORF’s All but the simplest genomes are works in progress. It is estimated that 80% of gene models have errors at present! Comparative genomics should help the process, as will sequencing of expressed sequence tags and other genomics projects Efficient implementation of a generalized pair hidden Markov model for comparative gene finding. W.H. Majoros, M. Pertea, and S.L. Salzberg. Bioinformatics 21:9 (2005),

Pfam Many others… HYSIELNASLLERGV … HLNIEDNPSCNAMGV … PLNIELNASLNEPGV … WERIELNASLNER--… HQRIEL--SLMMRG-… HLNIEDNPSCNAMGV … PLNIELNASLNEPGV… WERIELNASLNER--… HQRIEL--SLMMRG-… HYSIELNASLLERGV… HLNIEDNPSCNAMGV … WERIELNASLNER--… HQRIEL--SLMMRG-… HLNIEDNPSCNAMGV … PLNIELNASLNEPGV… WERIELNASLNER--… HQRIEL--SLMMRG-… HYSIELNASLLERGV… HLNIEDNPSCNAMGV … PLNIELNASLNEPGV… WERIELNASLNER--… HQRIELK-SLMMRG-… HYSIELNASLLERGV… HLNIEDNPSCNAMGV … PLNIELNASLNEPGV… WERIELNASLNER--… HQRIEL--SLMMRG-… The “sequence-space” of proteins Universe of all protein sequences PSI-BLAST HMM

PFAM “domains” Alex Bateman, Lachlan Coin, Richard Durbin, Robert D. Finn, Volker Hollich, Sam Griffiths-Jones, Ajay Khanna, Mhairi Marshall, Simon Moxon, Erik L. L. Sonnhammer, David J. Studholme, Corin Yeats and Sean R. Eddym Nucleic Acids Research(2004) Database Issue 32:D138-D141

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference Flow of information from DNA to functional understanding

X-ray Laboratory

Crystallography reveals locations of electron ‘clouds’ of the atoms: And the polypeptide chain can be traced through space

Scop Cath The “fold-space” of proteins Universe of all protein structures

Murzin et al.

Glimpes of the “fold space” of proteins Hou, Sims, Zhang, and Kim, PNAS 100:2386 (2003)

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference Flow of information from DNA to functional understanding

Connections between sequence and structure Universe of sequencesUniverse of structures

Connections between sequence and structure Universe of sequencesUniverse of structures ?

At what level of homology can one trust a structural inference? Redfern, Orengo et al., J. Chromatography B 815:97 (2005)

What is structural genomics? Experimental determination of key structures (target selection is a key part of the idea) Modeling of family members Inferring function (note “infer”) Making direct use of the new structures

Protein Sequences and Folds ~100,000 families of proteins that cannot be reliably modeled at present (modeling families: <30% identity over large fraction to a known structure) ~50% of all domain families can be assigned to a structure under CATH

Protein Structure Initiative (PSI) Mission Statement “To make the three-dimensional atomic level structures of most proteins easily available from knowledge of their corresponding DNA sequences.”

Genseration of new structures Chandonia and Brenner, Science 311:

Center for Eukaryotic Structural Genomics Exclusively eukaryotic targets 60% fold-space targets (emphasis on eukaryote-only families 20% disease relevant 20% outreach – targets from the community Overall goals are to reduce the costs of determining structures of proteins from eukaryotes by refining all steps in the pipeline Supported by National Institutes of Health John Markley- PI, George Phillips/Brian Fox Co-PI’s

University of Wisconsin’s Center for Eukaryotic Structural Genomics (~75 total, 3/4 unique)

How does one clone, express, purify, and solve structures not previously studied? An industry-style pipeline

Pipeline details: cell-based and cell-free protein production for X-ray and NMR Note: project involves sequencing, which aids gene modeling!

Sesame—integrated LIMS in use at CESG Open access to the public—structures, protocols, reagents, progress… Zolnai et al., J. Struct. Func. Genomics 4:11 (2003)

At1g18200 Mis-annotated prior to our work, but structure led to discovery of function.

>>Alignment of GalP_UDP_transf vs 1Z84:A|PDBID|CHAIN|SEQUENCE/ *->kkfsplDhvhrrynpLtlvwilVsphrakRPikqsqsLidlkkeLwq r p t +w+ sp+rakRP 1Z84:A|PDB 15 GDSVENQSPELRKDPVTNRWVIFSPARAKRP gavetpkvptdplhdp.dcysakLcpg atratgevNPdyest + ++k p+ p p++c+ c g r++ ++ P + 1Z84:A|PDB 46 -TDFKSKSPQNPNPKPsSCP---FCIGreqecapeLFRVP-DHDPNWKLR 90 yvLkspkkftndFyalseDnpyikvsvSNeaIaknplfqlksvrGhelci + +n ++als G +++ 1Z84:A|PDB 91 VI ENLYPALSRN---LETQ STQPETG--TSR 116 VI...CF......SKPehDptlpalakeeirevvdaWqlcteelGyegre +I + F++ +S P h+ l + i+ ++ a + + 1Z84:A|PDB 117 TIvgfGFhdvvieS-PVHSIQLSDIDPVGIGDILIAYKKRINQIA nhpayqnvqIFEmNkGaemGcsnpHPYaYFnEHGQvwatsfiP<-* h + + q+F N Ga G s H H Q a++ +P 1Z84:A|PDB 161 QHDSINYIQVFK-NQGASAGASMSHS------HSQMMALPVVP 196 Pfam B: 13 and 136 matches to #’s 7198 and

Blind prediction of structure: CASP and At5g18200

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference Flow of information from DNA to functional understanding

Function space of proteins KEGG = Kyoto Encyclopedia of Genes and Genomes The Gene Ontology project (GO) MetabolismCellular Processes Signal Processing Enzymes Don’t forget protein-protein interactions exist also!

At2g17340 Related to a human protein associated with Hallervorden-Spatz syndrome, a neurological disorder?

81 protein samples sent to Toronto: 8 solved CESG structures, 73 randomly chosen Generalized assays for: phosphatase, esterase, phospodiesterase, protease, amino acid dehydrogenase, alcohol dehydrogenase, organic acid dehydrogenase, amino acid oxidase, alcohol oxidase, organic acid oxidase, beta-lactamase, beta-galactosidase, arylsulfatase, lipase. Results: - Solid hits: 3 phosphatases, 5 esterases - Weaker hits: 9 more esterases, 6 phosphodiesterases - No hits: all others A. Yakuknin et al. Current Opinion in Chemical Biology, 8:42 (2004) Parallel Enzyme Activity Testing (Collaboration with University of Toronto)

Activity AssaySubstrateJR5670 Phosphodiesterasebis-pNPP0.016 DehydrogenaseAmino Acids0.032 DehydrogenaseAcids0.016 DehydrogenaseAlcohols0.022 DehydrogenaseAldehyde DehydrogenaseSugars0.003 Thioesterasepalmitoyl-CoA0.108 OxidaseNAD(P)H Ox ProteaseProtease Mix0.118 PhosphatasepNPP> 1 Target: At2g17340/JR5670 Absorbance >0.25 is a tentative signal, >0.5 is a strong signal. Initial Assay: Wide-spectrum

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference Flow of information from DNA to functional understanding

At2g17340 Enzyme of unknown specificity.

A functional annotation lesson

Functional Annotation by Inference From raw DNA sequences, one looks for genomic features such as promoters, alternative splicing of mRNAs, retrotransposons, pseudogenes, tandem duplications, synteny, and homology. It Is homology, both from sequence and from structure, that allow functional inferences to be made. Prosite, Dali, VAST, FFAS03 Some tool integrate knowledge from many sources into one place, acting a meta-servers of clues.

Connections between structure and function Universe of structures Universe of functions

Connections between structure and function Universe of structures Universe of functions Convergent evolution

Connections between structure and function Universe of structures Universe of functions Divergent evolution

At1g18200 Misleading annotation prior to our work, but structure led to discovery of function.

High-throughput DNA Sequencing Gene Model Functional Assignments Basic Understanding/ Applications (e.g. therapeutics) Structure Determination & Experimental Analysis Modeling & Inference Flow of information from DNA to functional understanding

Summary Structural genomics efforts are gaining momentum and helping to assign new functions to orfs and to fill in the space of all possible protein folds.

Administration Madison (Primm, Troestler, Markley, Phillips, Fox) Cloning/sequencing pipeline Madison (Wrobel, Fox) Expression pipeline Madison (Frederick, Fox, Riters) E. coli cell growth pipeline Madison (Sreenath, Burns, Seder, Fox) Cell-Free SystemMadison (Vinarov, Markley, Newman) Protein purification pipeline Madison (Vojtik, Phillips, Fox, Ellefson, Jeon) Mass spectrometry Madison (Aceti, Sabat, Sussman) Madison NMRFAM (Song, Tyler, Cornilescu, Markley) NMR spectroscopy Milwaukee MCW (Peterson, Volkman, Lytle) Crystallization / crystallography Madison (Bingman, Phillips, Bitto, Han, Bae, Meske) Argonne (Advanced Photon Source) BioinformaticsMadison (Bingman, Sun, Phillips, Wesenberg) Indianapolis (Dunker) Milwaukee MCW (Twigger, de la Cruz) Computational supportMadison (Bingman, Ramirez, Phillips) Sesame Madison (Zolnai, Markley, Lee) The Center for Eukaryotic Structural Genomics (supported by NIH GM64598 and GM074901)