Fabian Birzele Ludwig-Maximilians-University Munich Alternative Splicing and Protein Structure Evolution Fabian Birzele, Gergely Csaba and Ralf Zimmer, Nucleic Acids Research, 36 (2008),

2 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Introduction - Protein Structure Analysis Many more known structures than distinct protein structure topologies  cluster proteins on different levels of similarity (Topology, homology…)  SCOP and CATH Structure alignments reveal the insertions, deletions and mutations which have been tolerated by a structure family in evolution  “evolutionary isoforms” SCOP family d.9.1.1, Interleukin 8-like chemokines

3 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Introduction – Alternative splicing Alternative splicing assembles the exons of a gene in different ways 74% of all human genes are alternatively spliced (Johnson et al, Science 2003) Many examples where splicing isoforms carry out different functions Splicing increases the functional diversity of the proteome Protein1 Protein2 Function 1 Function 2 Introduction –Alternative splicing ?

4 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Alternative Splicing in the light of protein structures Very few experimental protein structures of splicing isoforms. Splicing changes structures only moderately: –Exposed, non-core elements (Wang et al., PNAS 2005) –Disordered (Romero et al., PNAS, 2006) –complete domains (e.g. Resch et al. J.Prot.Res. 2004) Splicing is deleterious for most protein structures –Often alters the hydrophobic core (Tress et al, PNAS, 2007 and Yura et al., Gene, 2006) Garcia et al., Nat. Struct. Biol., 2003  Tress et al: “… it seems unlikely that the spectrum of … enzymatic or structural function can be substantially extended through alternative splicing”

5 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Hypotheses Splicing is a major main contributor for functional diversity –Non-trivial splicing events can lead to proteins with well defined functions –Structures may tolerate large structural changes Tolerance against changes is linked to the evolutionary history of a protein fold –Evolutionary isoforms are a useful tool to understand splicing events –Alternative splicing is a useful tool to study protein structure evolution Alternative Splicing is a genetic mechanism to explore the protein fold space –Splice isoforms may adopt different folds in the protein fold space

6 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Methods and Data Splicing events annotated in Swissprot (high quality, manual annotations) Structures have been modeled very conservatively, 60% id, 75% coverage Multiple structure alignments of SCOP superfamilies  variable and conserved regions Results in 367 proteins and 488 additional isoforms Swissprot entry PDB structure Varsplic isoformsSCOP superfamily OK Non- trivial

7 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Distribution of splicing events OK (228) Non- trivial (260) Many splicing events are very complex on the structure level.  Are they functional?  What is their structure?

8 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Evolutionary isoforms help to understand splice isoforms

9 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Evolutionary isoforms MHC antigen recognition domain 1de4G, SCOP: d HFE_HUMAN, Isoform 2 Evolutionary isoform, same family Dimer (chain G and H) 1aqdH Evolutionary isoforms can help to understand the structure of splicing isoforms =

10 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Non-trivial isoforms can have well defined functions

11 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Functional evidence for non-trivial isoforms CC3 metastasis suppressor inducing apoptosis Splice variant TC3, 107 aa removed and 21 aa replaced, large scale event has anti-apoptotic function Reference: Whitman et al (1992) Mol. Cell. Biol. 20, remove replace

12 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Functional evidence for non-trivial isoforms LMPTP tyrosine phosphatase Removal of peripheral αβ-motif from β-sheet no phosphatase activity (active center removed) antogonist of native isoform –still binds substrates and regulators –But cannot dephosphorylate its targets Alter signaling pathways through non-trivial splicing events Reference: Tailor et al (1999) Eur. J. Biochem. 262,

13 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Functional evidence for non-trivial isoforms Comprehensive analysis of biological literature for all 260 non- trivial isoforms Only few studies analyzed isoforms on the protein and / or the function, but nevertheless: –17% (43) isoforms confirmed on the protein level –10% (26) isoforms with a well defined function –Often we find antagonistic functions  activator vs. repressor Those isoforms are interesting starting points for experimental structure determination

14 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Alternative Splicing and Fold Evolution

15 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Evolution of beta-propellers Beta-Propellers with different number of blades are known (4-8) but very low sequence identity and diverse functions  different folds in SCOP and CATH  Are they evolutionary related? Beta-propellers from different folds in SCOP are related on the sequence level (Chaudhuri, Söding and Lupas., Proteins 2008) Many known splicing events (60 in Swissprot, 50 in Human) where beta-propellers with different number of blades are generated from one gene  Different beta-propeller folds are explored from one gene  Connection between different propeller folds suggested by splicing

16 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Can splicing “tunnel” between folds? Different folds are not thought to be evolutionary related (or at least the signals are very weak) Many splicing events can not be explained within the own fold Can they be explained by structures from different folds?  Are those isoform structures more similar to proteins from a different fold than to their own fold? X X Splicing? X c.30 c.2 c.1

17 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 ?=?= Fold changing splicing event: c  c   splicing isoform c.2  c.30 SCOP apoptotic anti-apoptotic

18 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Can splicing “tunnel” between folds? In those cases we propose that the structure of the isoform adopts a fold different from the native one  traceable link between different folds Alternative splicing data provides evidence that it is possible to jump from one fold to the other  Splicing may be a genetic mechanism to explore the fold space X Splicing? Splicing! c.30 c.2 c.1

19 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 The ProSAS Database Comprehensive resource to analyze known splicing events in the context of protein structures. Events annotated in Swissprot and Ensembl (Human, Chimp, Mouse and Rat) ProSAS also allows for a structure- based analysis of experimental data like Affymetrix Exon Chips or Deep Sequencing –Those have the potential to verify or falsify our hypotheses

20 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Conclusion and Discussion Splicing is a major contributor to functional diversity, although the effects on the structure level are complex –Evolution makes use of the large plasticity of protein structures to generate functional and structural diversity via alternative splicing A structure‘s tolerance against changes is linked to its evolutionary history –Use evolutionary isoforms to understand alternative splicing –Use alternative splicing to understand structure evolution Isoforms may adopt folds different from the fold of the native isoform –Splicing may reveal novel links between protein folds Need for experimental structures and protein-level confirmations of isoforms

21 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Acknowledgements The U.S.Department of Energy (DOE) for the travel fellowship award: “Participant travel costs to present the project described was partly supported by Grant Number DE-FG02-06ER64270 from the U.S Department of Energy. The content is solely the responsibility of the author(s) and does not necessarily represent the official views of the Department of Energy.” My colleagues Gergely Csaba and Ralf Zimmer for their contribution to the paper and the talk The ProSAS-Team: Eva Hoffmann, Robert Küffner, Franziska Meier, Florian Oefinger, Christian Potthast You: for your attention

22 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Publications Fabian Birzele, Gergely Csaba and Ralf Zimmer, Alternative Splicing and Protein Structure Evolution, Nucleic Acids Research, 36 (2008), Fabian Birzele, Robert Küffner, Franziska Meier, Florian Oefinger, Christian Potthast and Ralf Zimmer, ProSAS: a database for analyzing alternative splicing in the context of protein structures, Nucleic Acids Research, 36 (2008), D63-D68

23 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Evolution of TIM-Barrels Have TIM-Barrels have evolved from gene fusion of half-barrels (Miles et al., Science 2000)? Are half-barrels generated by alternative splicing?

24 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 AUHM: Do events tunnel between different folds? c c SCOP

25 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Do events tunnel between different folds? SCOP c c Are there any connections between distinct folds in SCOP explored by alternative splicing? Search for “isoform structures” in a different fold based on structure similarity (TM-score), secondary structure fit and topology Splicing reveals possible links (“tunnels”) between different folds in SCOP for several examples

26 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Can splicing “tunnel” between folds? Different folds are not thought to be evolutionary related (or at least the signals are very weak) Many splicing events can not be explained within the own fold  Are those isoform structures more similar to proteins from a different fold than to their own fold? Alternative splicing data provides evidence that it is possible to jump from one fold to the other  Splicing may be a genetic mechanism to explore the fold space X X Splicing? X Splicing!

27 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Superfamily b ok3_A, b , CYP10_CAAEL 1x7f_A, b

28 Alternative splicing and protein structure evolution Fabian Birzele, LMU Institut für Informatik, Lehrstuhl für Praktische Informatik und Bioinformatik, ISMB 2008 Functional evidence for non-trivial isoforms Caspase 9: induces apoptosis Splice variant Caspase 9b has more than 50% of the residues removed Nevertheless, variant is an apoptosis inhibitor Reference: Srinivasula et al (1999) Cancer Res. 59,