1. (A) Protein-protein interaction and (B) Nucleic Acid Structure Lecture 19: Introduction to Bioinformatics C E N T R F O R I N T E G R A T I V E B I O I N F O R M A T I C S V U E

2. Lecture 19A: Protein-protein interactions Complexity: –Multibody interaction Diversity: –Various interaction types Specificity: –Complementarity in shape and binding properties

3. PPI Characteristics Universal –Cell functionality based on protein-protein interactions Cyto-skeleton Ribosome RNA polymerase Numerous –Yeast: ~6.000 proteins at least 3 interactions each  ~18.000 interactions –Human: estimated ~100.000 interactions Network –simplest: homodimer (two) –common: hetero-oligomer (more) –holistic: protein network (all)

4. Interface Area Contact area –usually >1100 Å 2 –each partner >550 Å 2 each partner loses ~800 Å 2 of solvent accessible surface area –~20 amino acids lose ~40 Å 2 –~100-200 J per Å 2 Average buried accessible surface area: –12% for dimers –17% for trimers –21% for tetramers 83-84% of all interfaces are flat Secondary structure: –50%  -helix –20%  -sheet –20% coil –10% mixed Less hydrophobic than core, more hydrophobic than exterior

5. Complexation Reaction A + B  AB –K a = [AB]/[A][B]  association –K d = [A][B]/[AB]  dissociation

6. Experimental Methods for determining PPI 2D (poly-acrylamide) gel electrophoresis  mass spectrometry Liquid chromatography –e.g. gel permeation chromatography Binding study with one immobilized partner –e.g. surface plasmon resonance In vivo by two-hybrid systems or FRET Binding constants by ultra-centrifugation, micro- calorimetry or competition Experiments with labelled ligand –e.g. fluorescence, radioactivity Role of individual amino acids by site directed mutagenesis Structural studies –e.g. NMR or X-ray

7. PPI Network http://www.phy.auckland.ac.nz/staff/prw/biocomplexity/protein_network.htm

8. Binding vs. Localization Obligate oligomers Non-obligate weak transient Non-obligate triggered transient e.g. GTPPO 4 - Non-obligate co-localised e.g. in membrane Non-obligate permanent e.g. antibody-antigen strong weak co-expressed and at same place different places

9. Some terminology Transient interactions: –Associate and dissociate in vivo Weak transient: –dynamic oligomeric equilibrium Strong transient: –require a molecular trigger to shift the equilibrium Obligate PPI: –protomers no stable structures on their own (i.e. they need to interact in complexes) –(functionally obligate)

10. Analysis of 122 Homodimers 70 interfaces single patched 35 have two patches 17 have three or more

11. Interfaces ~30% polar ~70% non-polar

12. Interface Rim is water accessible rim interface

13. Interface composition Composition of interface essentially the same as core But % surface area can be quite different! = different surface/interface areas

14. Some preferences prefer avoid

15. Ribosome structure In the nucleolus, ribosomal RNA is transcribed, processed, and assembled with ribosomal proteins to produce ribosomal subunits At least 40 ribosomes must be made every second in a yeast cell with a 90-min generation time (Tollervey et al. 1991). On average, this represents the nuclear import of 3100 ribosomal proteins every second and the export of 80 ribosomal subunits out of the nucleus every second. Thus, a significant fraction of nuclear trafficking is used in the production of ribosomes. Ribosomes are made of a small and a large subunit Large (1) and small (2) subunit fit together (note this figure mislabels angstroms as nanometers)

16. Ribosome structure The ribosomal subunits of prokaryotes and eukaryotes are quite similar but display some important differences. Prokaryotes have 70S ribosomes, each consisting of a (small) 30S and a (large) 50S subunit, whereas eukaryotes have 80S ribosomes, each consisting of a (small) 40S and a bound (large) 60S subunit. However, the ribosomes found in chloroplasts and mitochondria of eukaryotes are 70S, this being but one of the observations supporting the endosymbiotic theory. "S" means Svedberg units, a measure of the rate of sedimentation of a particle in a centrifuge, where the sedimentation rate is associated with the size of the particle. Note that Svedberg units are not additive. Each subunit consists of one or two very large RNA molecules (known as ribosomal RNA or rRNA) and multiple smaller protein molecules. Crystallographic work has shown that there are no ribosomal proteins close to the reaction site for polypeptide synthesis. This suggests that the protein components of ribosomes act as a scaffold that may enhance the ability of rRNA to synthesise protein rather than directly participating in catalysis. The differences between the prokaryotic and eukaryotic ribosomes are exploited by humans since the 70S ribosomes are vulnerable to some antibiotics that the 80S ribosomes are not. This helps pharmaceutical companies create drugs that can destroy a bacterial infection without harming the animal/human host's cells!

17. 70S structure at 5.5 Å (Noller et al. Science 2001)

18. 70S structure

19. 30S-50S interface Overall buried surface area ~8500 Å 2 < 37.5 Å 2 37.5 Å 2 – 75 Å 2 > 75 Å 2

20. Protein-nucleic acid Interactions

21. Interactions in the Ribosome

22. Docking - ZDOCK Protein-protein docking –3-dimensional (3D) structure of protein complex –starting from 3D structures of receptor and ligand Rigid-body docking algorithm (ZDOCK) –pairwise shape complementarity function –all possible binding modes –using Fast Fourier Transform algorithm Refinement algorithm (RDOCK) –Take top 2000 predicted structures from ZDOCK (RDOCK is too computer intensive to refine very many possible dockings) –three-stage energy minimization –electrostatic and desolvation energies molecular mechanical software (CHARMM) statistical energy method (Atomic Contact Energy) 49 non-redundant unbound test cases: –near-native structure (<2.5Å) on top for 37% test cases for 49% within top 4

23. Protein-protein docking Finding correct surface match Systematic search: –2 times 3D space! Define functions: –‘1’ on surface –‘  ’ or ‘  ’ inside –‘0’ outside  

24. Protein-protein docking Correlation function: C  = 1/N 3  o  p  q exp[2  i(o  + p  + q  )/N] C o,p,q

25. Docking Programs ZDOCK, RDOCK AutoDock Bielefeld Protein Docking DOCK DOT FTDock, RPScore and MultiDock GRAMM Hex 3.0 ICM Protein-Protein docking (Abagyan group, currently the best) KORDO MolFit MPI Protein Docking Nussinov-Wolfson Structural Bioinformatics Group …

26. Docking Programs Issues: Rigid structures or made flexible? –Side-chains –Main-chains Full atomic detail or simplified models? Docking energy functions (purpose built force fields)

27. Docking example: antibody HyHEL-63 (cyan) complexed with Hen Egg White Lysozyme The X-ray structure of the antibody HyHEL-63 (cyan) uncomplexed and complexed with Hen Egg White Lysozyme (yellow) has shown that there are small but significant, local conformational changes in the antibody paratope on binding. The structure also reveals that most of the charged epitope residues face the antibody. Details are in Li YL, Li HM, Smith-Gill SJ and Mariuzza RA (2000) The conformations of the X-ray structure Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63. Biochemistry 39: 6296-6309. Salt links and electrostatic interactions provide much of the free energy of binding. Most of the charged residues face in interface in the X-ray structure. The importance of the salt link between Lys97 of HEL and Asp27 of the antibody heavy chain is revealed by molecular dynamics simulations. After 1NSec of MD simulation at 100°C the overall conformation of the complex has changed, but the salt link persists. Details are described in Sinha N and Smith-Gill SJ (2002) Electrostatics in protein binding and function. Current Protein & Peptide Science 3: 601-614.

28. Introduction to Bioinformatics Lecture 19B: Nucleic acid structure

29. Nucleic Acid Basics Nucleic Acids Are Polymers Each Monomer Consists of Three Moieties: Nucleotide A Base + A Ribose Sugar + A Phosphate Nucleoside A Base Can be One of the Five Rings:

30. Pyrimidines Purines Pyrimidines and Purines can Base-Pair (Watson-Crick Pairs)

32. Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent of their sequences. There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries. The double helical structure is essential to the coding function of DNA. Watson (biologist) and Crick (physicist) first discovered the double helix structure in 1953 by X-ray crystallography. RNA, on the other hand, can have as diverse structures as proteins, as well as simple double helix of type A. The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis). In fact, some viruses encode their genetic materials by RNA (retrovirus)

33. Forces That Stabilize Nucleic Acid Double Helix There are two major forces that contribute to stability of helix formation –Hydrogen bonding in base-pairing –Hydrophobic interactions in base stacking 5’ 3’ Same strand stacking cross-strand stacking

34. Types of DNA Double Helix Type A: major conformation of RNA, minor conformation of DNA; Type B: major conformation of DNA; Type Z: minor conformation of DNA 5’ 3’ 5’ 3’ 5’ 3’ AB Z Narrow tight Wide Less tight Left-handed Least tight

35. Three Dimensional Structures of Double Helices A-DNA A-RNA Major Groove Minor Groove A-DNA

36. Secondary Structures of Nucleic Acids DNA is primarily in duplex form. RNA is normally single stranded which can have a diverse form of secondary structures other than duplex.

37. More Secondary Structures of Nucleic Acids Pseudoknots: Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press.

38. 3D Structures of RNA: Transfer RNA Structures Anticodon Stem D Loop T  C Loop Variable loop Anticodon Loop Secondary Structure of tRNA Tertiary Structure of tRNA Gm, Cm, etc., are modified bases

39. Ban et al., Science 289 (905-920), 2000 Secondary Structure Of large ribosomal RNA Tertiary Structure Of large ribosome subunit 3D Structures of RNA: Ribosomal RNA Structures rRNA Secondary Structure Based on Phylogenetic Data

40. Central Dogma of Molecular Biology Replication DNA Transcription mRNA Translation Protein Transcription is carried out by RNA polymerase (II) Translation is performed on ribosomes Replication is carried out by DNA polymerase Reverse transcriptase copies RNA into DNA Transcription + Translation = Expression

41. But DNA can also be transcribed into non- coding RNA …  tRNA (transfer): transfer of amino acids to the ribosome during protein synthesis.  rRNA (ribosomal): essential component of the ribosomes (complex with rProteins).  snRNA (small nuclear): mainly involved in RNA-splicing (removal of introns). snRNPs.  snoRNA (small nucleolar): involved in chemical modifications of ribosomal RNAs and other RNA genes. snoRNPs.  SRP RNA (signal recognition particle): forms RNA-protein complex involved in mRNA secretion.  Further: microRNA,,eRNA, gRNA, tmRNA etc.

42. Eukaryotes have spliced genes …  Promoter: involved in transcription initiation (TF/RNApol-binding sites)  TSS: transcription start site  UTRs: un-translated regions (important for translational control)  Exons will be spliced together by removal of the Introns  Poly-adenylation site important for transcription termination (but also: mRNA stability, export mRNA from nucleus etc.)

43. DNA makes mRNA makes Protein

44. Some facts about human genes  There are about 20.000 – 25.000 genes in the human genome (~ 3% of the genome)  Average gene length is ~ 8.000 bp  Average of 5-6 exons per gene  Average exon length is ~ 200 bp  Average intron length is ~ 2000 bp  8% of the genes have a single exon  Some exons can be as small as 1 or 3 bp

45. DMD: the largest known human gene  The largest known human gene is DMD, the gene that encodes dystrophin: ~ 2.4 milion bp over 79 exons  X-linked recessive disease (affects boys)  Two variants: Duchenne-type (DMD) and Becker-type (BMD)  Duchenne-type: more severe, frameshift-mutations Becker-type: milder phenotype, “in frame”- mutations Posture changes during progression of Duchenne muscular dystrophy

46. Nucleic acid basics  Nucleic acids are polymers  Each monomer consists of 3 moieties nucleoside nucleotide

47. Nucleic acid basics (2)  A base can be of 5 rings  Purines and Pyrimidines can base-pair (Watson- Crick pairs) Watson and Crick, 1953

48. Nucleic acid as hetero-polymers  Nucleosides, nucleotides (Ribose sugar, RNA precursor) (2’-deoxy ribose sugar, DNA precursor) (2’-deoxy thymidine tri- phosphate, nucleotide)  DNA and RNA strands REMEMBER: DNA =deoxyribonucleotides; RNA =ribonucleotides (OH-groups at the 2’ position) Note the directionality of DNA (5’-3’ & 3’-5’) or RNA (5’-3’) DNA = A, G, C, T ; RNA = A, G, C, U

49. So … DNARNA

50. Stability of base-pairing  C-G base pairing is more stable than A-T (A-U) base pairing (why?)  3 rd codon position has freedom to evolve (synonymous mutations)  Species can therefore optimise their G-C content (e.g. thermophiles are GC rich) (consequences for codon use?) Thermocrinis ruber, heat-loving bacteria

51. DNA compositional biases  Base compositions of genomes: G+C (and therefore also A+T) content varies between different genomes  The GC-content is sometimes used to classify organism in taxonomy  High G+C content bacteria: Actinobacteria e.g. in Streptomyces coelicolor it is 72% Low G+C content: Plasmodium falciparum (~20%)  Other examples: Saccharomyces cerevisiae (yeast)38% Arabidopsis thaliana (plant)36% Escherichia coli (bacteria)50%

52. Let’s return to DNA and RNA structure …  Unlike three dimensional structures of proteins, DNA molecules assume simple double helical structures independent on their sequences.  There are three kinds of double helices that have been observed in DNA: type A, type B, and type Z, which differ in their geometries.  RNA on the other hand, can have as diverse structures as proteins, as well as simple double helix of type A.  The ability of being both informational and diverse in structure suggests that RNA was the prebiotic molecule that could function in both replication and catalysis (The RNA World Hypothesis).  In fact, some viruses encode their genetic materials by RNA (retrovirus)

53. Three dimensional structures of double helices Side view: A-DNA, B-DNA, Z-DNA Top view: A-DNA, B-DNA, Z-DNA Space-filling models of A, B and Z- DNA

54. Major and minor grooves

55. Forces that stabilize nucleic acid double helix  There are two major forces that contribute to stability of helix formation: Hydrogen bonding in base-pairing Hydrophobic interactions in base stacking 5’ 3’ Same strand stacking cross-strand stacking

56. Types of DNA double helix  Type A major conformation RNA minor conformation DNA Right-handed helix Short and broad  Type B major conformation DNA Right-handed helix Long and thin  Type Z minor conformation DNA Left-handed helix Longer and thinner

57. Secondary structures of Nucleic acids  DNA is primarily in duplex form  RNA is normally single stranded which can have a diverse form of secondary structures other than duplex.

58. Non B-DNA Secondary structures  Cruciform DNA  Triple helical DNA  Slipped DNA Hoogsteen basepairs Source: Van Dongen et al. (1999), Nature Structural Biology 6, 854 - 859

59. More Secondary structures  RNA pseudoknots  Cloverleaf rRNA structure Source: Cornelis W. A. Pleij in Gesteland, R. F. and Atkins, J. F. (1993) THE RNA WORLD. Cold Spring Harbor Laboratory Press. 16S rRNA Secondary Structure Based on Phylogenetic Data

60. 3D structures of RNA : transfer-RNA structures  Secondary structure of tRNA (cloverleaf)  Tertiary structure of tRNA

61. 3D structures of RNA : ribosomal-RNA structures  Secondary structure of large rRNA (16S)  Tertiary structure of large rRNA subunit Ban et al., Science 289 (905-920), 2000

62. 3D structures of RNA : Catalytic RNA  Secondary structure of self-splicing RNA  Tertiary structure of self-splicing RNA

63. Some structural rules …  Base-pairing is stabilizing  Un-paired sections (loops) destabilize  3D conformation with interactions makes up for this

64. Final notes  Sense/anti-sense RNA antisense RNA blocks translation through hybridization with coding strand Example. Tomatoes synthesize ethylene in order to ripe. Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production  tomatoes make only 10% of normal enzyme amount.  Sense/anti-sense peptides Have been therapeutically used Especially in cancer and anti-viral therapy  Sense/anti-sense proteins Does it make (anti)sense? Codons for hydrophilic and hydrophobic amino acids on the sense strand may sometimes be complemented, in frame, by codons for hydrophobic and hydrophilic amino acids on the antisense strand. Furthermore, antisense proteins may sometimes interact with high specificity with the corresponding sense proteins… BUT VERY RARE: HIGHLY CONSERVED CODON BIAS