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V4: Area 3 – interaction networks Area 1: Systems of coupled differential equations (T. Geyer) Area 2: Metabolic networks - flux balance analysis - MILP - elementary flux modes, linear algebra background - apply software FluxAnalyzer to test systems Area 3: Graph networks – interaction networks - network of proline-rich sequences and adaptor domains - apply software Cytoscape to test systems Area 4: Spatial modelling of cellular systems (T. Geyer) 4. Lecture SS 20005 Cell Simulations 1 SH3 as a structural motif in SRC tyrosine kinase Domains that bind proline-rich motifs are critical to the assembly of many intracellular signaling complexes and pathways. The importance of proline-rich motifs in biology is highlighted by the finding that proline-rich regions are the most common sequence motif in the Drosophila genome and the second most common in the Caenorhabditis elegans genome. The number of defined protein domains that recognize proline-rich motifs has expanded considerably in recent years to include such common motifs as Src homology 3 (SH3), WW (named for a conserved Trp-Trp motif), and Enabled/VASP homology (EVH1, also known as WASP homology 1 or WH1) domains, as well as other proline-binding domains. The number of domains in an organism roughly corresponds to its perceived complexity (Table 1). Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 2 First crystal structures of SH3 First X-ray structure of a SH3 domain in 1992. Musacchio,A., Noble, M., Pauptit, R.,Wierenga, R. and Saraste, M. (1992):Crystal structure of a Src-homology 3 (SH3) domain. Nature 359, 851-855 First X-ray structure of a complex of SH3 with proline rich ligand in 1994: Musacchio,A., Saraste, M. andWilmanns, M. (1994): High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nature Struct. Biol. 1, 546-551 4. Lecture SS 20005 Cell Simulations 3 Function of proline recognition domains Proline recognition domains are usually found in the context of larger multidomain signaling proteins. Their binding events often direct the assembly and targeting of protein complexes involved in - cell growth - cytoskeletal rearrangements - transcription - postsynaptic signaling - and other key cellular processes In addition, these interactions can play a regulatory role, often through autoinhibitory interactions that are alleviated by competing binding events. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 4 Example: negative regulation of T-cell receptor by adaptor domains Examples of negative regulation by adaptor molecules and adaptor domains are depicted. a Allosteric inhibition by the adaptor domains of SRC-family kinases. The SRC-homology 2 (SH2) domain of SRCfamily kinases binds to the carboxyterminal phosphotyrosine residue, thereby restricting substrate accessibility and kinase activity. The SH3 domain has also been shown to regulate SRC kinase activity through intramolecular interactions that create an inducible 'snap lock', which is dependent on interdomain hinge regions as well. On dephosphorylation of the C-terminal tyrosine by the CD45 phosphatase, the adaptor domains are released and result in activation of the kinase. b Recruitment of negative effector molecules to their substrates. In unstimulated T cells, raft-associated PAG/CBP is constitutively tyrosine phosphorylated and associates with the SH2 domain of CSK, bringing CSK into close proximity to its substrates (SRC-family PTKs) at the plasma membrane. Following TCR stimulation, PAG/CBP is dephosphorylated, resulting in the release of CSK from the membrane and relieving SRC-family kinases from CSK phosphorylation-mediated inhibition. Evidence also indicates that PAG/CBP might also regulate CSK activity independently of its ability to recruit CSK to lipid rafts. Nature Reviews Immunology 1; 95-107 (2001) 4. Lecture SS 20005 Cell Simulations 5 SH3 as a structural motif in SRC tyrosine kinase http://jkweb.berkeley.edu/external/pdb/1997/hck/hck.html 4. Lecture SS 20005 Cell Simulations 6 A proline-driven conformational switch within the Itk SH2 domain NMR structures of the cis and trans Itk SH2 conformers. a, Stereo view of 20 low energy structures of the cis (coral) and trans (turquoise) conformations of the Itk SH2 domain. Backbone heavy atoms within the secondary structural elements over the entire sequence were used for superpositions. b, Ribbon diagrams of the energy minimized average structures of the cis (left) and trans (right) conformers. Secondary structural elements and ligand-binding pockets are labeled in (a,b) according to standard nomenclature for SH2 domains8. Pro 287 is labeled in each structure. c, Sequence of the Itk SH2 domain and sequence alignment of the CD loop regions in the SH2 domains of several tyrosine kinases. The residues that give rise to nondegenerate chemical shifts2 are bold and underlined, and Pro 287 is labeled. e, Overlay of the energy minimized average structures of the cis (coral) and trans (turquoise) conformers. Expanded views of the CD loop (left), the central -sheet (right) and the BG loop regions (middle) are shown. Mallis, Brazin, Fulton, Andreotti, Structural characterization of a proline-driven conformational switch within the Itk SH2 domain, Nat. Struct. Biol. 9, 900 - 905 (2002) 4. Lecture SS 20005 Cell Simulations 7 Structural differences between cis and trans isomers Structural differences between the cis and trans Itk SH2 domain provide a basis for conformer-specific binding to the Itk SH3 domain. a, A backbone ribbon representation of the Itk cis SH2 domain with the Itk polyproline peptide (KPLPPTP shown in white) superimposed on the structure. The polyproline peptide residues are labeled using the one letter amino acid code and are numbered consecutively. In previously determined peptide–SH3 structures10, 11, Lys 1 (K1), Leu 3 (L3), Pro 4 (P4), Thr 6 (T6) and Pro 7 (P7) directly contact the SH3-binding pocket, whereas Pro 2 (P2) and Pro 5 (P5) do not. SH2 domain residues that are involved in SH3 binding (as determined by chemical shift mapping) are highlighted in yellow and labeled with bold-letter font. Putative correlations between SH2 residues and the canonical polyproline peptide are as follows: Arg 332-Lys 1, Val 330-Leu 3, Thr 279-Pro 4, Cys 288-Thr 6 and Ile 282-Pro 7. This model was arrived at by initial superposition of the basic peptide residue Lys 1 with Arg 332 of the SH2 domain. This assignment is based on the large chemical shift perturbation observed for Arg 332 upon addition of SH3 ( 15N = 0.407 p.p.m. and 1H = 0.196 p.p.m.) and the observation that previously determined SH3–ligand complexes, combined with mutational analyses, have shown that a stabilizing interaction involving a basic amino acid side chain and a conserved acidic site within the SH3 domain is required for SH3 ligand binding32. Subsequently, using Arg 332 as an anchor, the polyproline peptide structure was rotated over the surface of the cis SH2 domain to assess whether the cis SH2 residues that mediate binding to the SH3 domain may be similar in geometric arrangement and chemical nature to the polyproline peptide side chains known to contact the SH3 binding surface in SH3– peptide complexes11, 32. b, Isomerization to the trans SH2 structure disrupts the putative binding site on the cis SH2 domain, which is consistent with the inability of trans SH2 to bind to the SH3 domain. Residue coloring and labeling same as shown in (a). Mallis, Brazin, Fulton, Andreotti, Structural characterization of a proline-driven conformational switch within the Itk SH2 domain, Nat. Struct. Biol. 9, 900 - 905 (2002) 4. Lecture SS 20005 Cell Simulations 8 Why are proline-rich sequences special? Repetitive proline-rich sequences are found in many proteins and in many cases are thought to function as docking sites for signaling modules. Why might proline be singled out for recognition by so many key protein-protein interaction modules? Several features of proline distinguish it from the other 19 naturally occurring amino acids (Fig. 1A): - the unusual shape of its pyrrolidine ring - the conformational constraints on its dihedral angles imposed by this cyclic side chain - its resulting secondary structural preferences - its substituted amide nitrogen, - and the relative stability of the cis isomer in a peptide bond. Each recognition domain exploits some combination of these distinctive features of proline in order to achieve specific binding to proline-rich regions. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 9 Polyproline type II (PPII) helices One feature of proline-rich motifs that is frequently used in binding to signaling domains is their propensity to form a polyproline type II (PPII) helix. The PPII helix is an extended left-handed helical structure with three residues per turn and an overall shape resembling a triangular prism (Fig. 1B). A combination of steric and hydrogen-bonding properties of proline-rich motifs is thought to contribute to its preference for this unusual secondary structure. Two features of the PPII helix make it a useful recognition motif: First, in this structure both the side chains and the backbone carbonyls point out from the helical axis into solution at regular intervals (Fig. 1B). The lack of intramolecular hydrogen bonds in the PPII structure, due largely to the absence of a backbone hydrogen-bond donor on proline, leaves these carbonyls free to participate in intermolecular hydrogen bonds. Thus, both side chains and carbonyls can easily be “read” by interacting proteins. Second, because the backbone conformation in a PPII helix is already restricted, the entropic cost of binding is reduced. Nearly all of the domains described here bind their ligands in a PPII conformation. Many of the interactions with the PPII helical ligand involve aromatic residues. The planar structure of aromatic side chains appears to be highly complementary to the ridges and grooves presented on the PPII helix surface. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 10 Properties of PPII helices (B) Schematic and structural representation of a PPII helix. The helix has twofold pseudosymmetry: A rotation of 180° about a vertical axis leaves the proline rings and the carbonyl oxygens at approximately the same position. The Protein Data Bank (PDB) accession code for the poly-(l)proline structure shown is 1CF0. (C) A view down the axis of the PPII helix highlighting the position of the carbons in the xP dipeptide. In the “x” position that requires C-substitution (blue), the primary recognition element is the β carbon, whereas in the “P” position that requires N-substitution (red), the primary recognition element is the δ carbon that is unique to proline. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 11 Polyproline type II (PPII) helices One interesting structural feature of the PPII helix is that it has twofold rotational pseudosymmetry: Side chains and backbone carbonyls are displayed with similar spacing in either of the two N- to C-terminal orientations (Fig. 1B). This feature may explain why many proline-binding domains are observed to bind ligands in two possible orientations, a property unique among characterized peptide recognition modules. In principle, this orientational flexibility could play an important role in domain function. For example, one could imagine a complex in which binding in one orientation could be activating, whereas binding in the opposite orientation could be inhibitory. However, such an orientational switching role has not been demonstrated. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 12 Polyproline type II (PPII) helices Another unique property of proline is that it is the only naturally occurring Nsubstituted amino acid. Proteins that recognize the δ carbon on the substituted amide nitrogen (Fig. 1A) within the context of the otherwise standard peptide backbone can select precisely for proline at a given position without making extended contacts with the rest of the side chain (Fig. 1C). Thus, sequencespecific recognition can be achieved without requiring a particularly high-affinity interaction. Interactions that are specific and low-affinity can be quite useful in intracellular signaling environments where rapidly reversible interactions may be required. Among proline-binding domains, this phenomenon has been best characterized for SH3 domains, in which required prolines can be replaced without a significant loss in binding affinity by a number of nonnatural N-substituted amino acids that do not resemble proline. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 13 Polyproline type II (PPII) helices Proline also stands out from other natural amino acids in its ability to exist stably as a cis isomer about the peptide bond. In an unfolded chain, proline residues adopt the cis conformation with a probability of ~20% as compared to negligible amounts for the other amino acids. Moreover, the kinetic barrier for cis-trans isomerization is higher for proline than for the other amino acids and is even the rate-limiting step in the folding of certain proteins. In principle, recognition of cis proline moieties could be a useful way of achieving regulation, potentially even with some degree of kinetic control. However, none of the major proline recognition modules discussed here are known to exploit recognition of cis isomers. Still, the intriguing possibility remains that cis-trans isomerization could provide a mechanism to modulate such recognition events. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 14 Properties of proline Thus, many chemical properties of proline distinguish it from the other 19 naturally occurring amino acids, and proline recognition domains exploit several of these properties. If a recognition event involves a property of proline that is sufficiently distinct among the natural set of 20 amino acids, the interaction does not have to be of particularly high affinity to be selective. The benefits of weak, but specific, interactions in intracellular signaling pathways may help explain the abundance of proline-based recognition motifs. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 15 Functional roles of SH3 domains (A) Assembly role of SH3 domains. Growth factor stimulation leads to the activation of receptor tyrosine kinases and to the phosphorylation of the receptor tail, of related adaptor proteins (not shown), or of both. The resultant phosphotyrosines form docking sites for the adaptor protein Grb2 (through its SH2 domain). The Grb2 SH3 domains bind proline-rich motifs in SOS, the guanine nucleotide exchange factor for Ras, recruiting SOS to the membrane and colocalizing it with Ras. The resultant stimulation of Ras activates a MAPK cascade, leading to cell growth and differentiation. (B) Regulatory role of SH3 domains. Intramolecular interactions of the SH2 and SH3 domains of Src kinases hold their kinase domains in an inactive conformation. These autoinhibitory interactions can be disrupted by external SH2 and SH3 ligands, yielding spatial and temporal control of kinase activation. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 16 Functional roles of SH3 domains Structure and binding mechanism of SH3 domains. The structure of the Sem5 SH3 domain in complex with a proline-rich ligand is shown. A cartoon of the proline-binding surface of these domains docked with a ligand, showing the general mechanism of recognition, is shown below. The core recognition surface has two xP binding grooves formed by aromatic amino acids, shown in yellow, and the adjacent, less conserved specificity pockets are designated in green. The PDB accession code for this structure is 1SEM. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 17 Structure and binding mechanism of WW domains The structure of the dystrophin WW domain in complex with a proline-rich ligand is shown. A cartoon of the proline-binding surface of these domains docked with a ligand, showing the general mechanism of recognition, is shown on the right. The core recognition surface has one xP binding groove formed by aromatic amino acids (yellow) and adjacent, less conserved specificity pockets (green). The PDB accession code for this structure is 1EG4 Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 18 Structure and binding mechanism of EVH1 domains A representative structure of the Mena EVH1 domain in complex with a peptide ligand is shown. Below is a schematic of the recognition mechanism showing the apex of the PPII helix fitting into an aromatic-rich wedge at the binding surface. Although a conserved set of aromatic residues (yellow) also contacts the PPII ligand, the manner in which the PPII helix docks against the domain surface differs from that observed in most other proline-binding domains discussed here. The PDB accession code for this structure is 1EVH. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 19 Structure and binding mechanism of a GYF domain The structure of the CD2BP2 GYF domain in complex with a proline-rich ligand is shown. A cartoon of the proline-binding surface of these domains docked with a ligand is shown below. The core recognition surface has one xP binding groove formed by aromatic amino acids (yellow) and adjacent, less conserved specificity pockets (green). The PDB accession code for this structure is 1L2Z Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 20 Mechanisms for enhancing the specificity Potential mechanisms for enhancing the specificity of proline-binding domains. One means of increasing specificity in proline-mediated interactions is by extending the interaction surface with the peptide to include residues beyond the proline-rich core. Another mechanism is to include a nearby sequence on the ligand that interacts with another binding module in the same complex as the proline recognition module. A third mechanism adds a separate recognition surface onto the proline recognition domain that recognizes a distinct peptide. Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8. 4. Lecture SS 20005 Cell Simulations 21 Identification of a novel “register-shifted” binding mode NMR structure of GYF domain with wildtype peptide. The GYF domain is represented by its molecular surface; the peptide atoms are drawn as sticks. Residues forming the binding pocket are coloured in dark grey and labelled by their one-letter codes and sequence numbers. Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 22 What is the conformation of the unbound peptide Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 23 Study conformation of unbound peptide Evolution of the backbone dihedral angles (black: Phi angles; red: Psi angles) during the MD simulation of the wild-type peptide (a) and the mutant peptide (b). Ideal values of the dihedral angles are shown in solid lines (blue: Phi angles; green: Psi angles). Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 24 Unbound peptides have PPII helical conformation Superposition of the representative conformations of simulations of unbound peptides (from left to right: WT, WTE, G8W and H9M) onto the bound peptide in the NMR structure. Representative conformations are colored in black while the bound peptide in the NMR structure is shown in grey. Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 25 Which residues are crucial for binding? WT A C D E F G H I K L M N P Q R S T V W Y D E F G H I K L M N P Q R S T V W Y S H R P P P P G H R V WT A C S H R P P P P W H R V Conclusion: Two central prolines are critical and the following glycine. But can this glycine be mutated to Trp? Substitution analysis of the SHRPPPPGHR peptide binding to the GYF domain. All single substitution analogues of the peptide were synthesized on a cellulose membrane. The single letter code above each column marks the amino acid that replaces the corresponding wild-type residue, while the row defines the position of the substitution within the peptide. Spots in the most left column (WT) have identical sequences and represent the wild type peptide. The membrane was incubated with a GST-GYF construct of CD2BP2. Bound protein was detected with an anti-GST primary antibody and a horse-radish peroxidase coupled secondary antibody. The relative spot intensities correlate qualitatively with the binding affinities Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 26 Binding analysis of Trp-peptide mutant Binding analysis of the CD2BP2-GYF domain to the peptide SHRPPPPWHRV in comparison to the wild-type peptide SHRPPPPGHRV by NMR. (a) The sum of the weighted geometrical differences of the chemical shifts (Geometric sum of chemical shift changes) for assigned peaks, which could be identified at all applied peptide concentrations is plotted against the concentration of the peptide. (b) Mapping of the binding site of SHRPPPPGHRV and SHRPPPPWHRV peptides onto the CD2BP2GYF domain. Overlay of HSQC spectra of GYF domain alone (green) and GYF-domain in the presence of a 10-fold excess of the wild-type peptide SHRPPPPGHRV (blue) or the mutant peptide SHRPPPPWHRV (red), respectively. Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 27 MD simulation of GYF:domain complexes Comparison of the binding interfaces of the GYF domain (NMR and simulation) for the wild-type complex (above) and of the H9M mutant (below). The GYF domain is represented by its molecular surface and coloured by position (from orange to deep blue: completely buried to completely exposed); the peptide atoms are drawn as sticks and coloured according to their appearance in sequence. Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 28 Trp-peptide mutant shows “register shift” (a) Superposition of the two binding modes found in the simulation of the G8W mutant complex (starting from the docking results). The two conformations of the peptide are drawn as sticks (blue: mode 1, red: mode 2, pink: Pro6 and Pro7 in mode 1, yellow: Pro6 and Pro7 in mode 2). (b) Binding mode of the G8R mutant complex (representative conformation of the simulation). The peptide atoms are represented by sticks and coloured according to their sequence number. In (a) and (b), the GYF domain is represented by its molecular surface and coloured by position (from orange to deep blue: completely buried to completely exposed) and Pro6 and Pro7 are labelled by their one-letter codes and sequence numbers. Mode 2 is labelled as “(alt)”. (c) Superposition of the representative conformations of the five simulations of wild type GYF complex starting from the alternative binding mode. Pro6 and Pro7 are represented by sticks and are labelled by their one-letter codes and sequence numbers. Pro6 is coloured in light grey and Pro7 is coloured in dark grey. (d) The translation and rotation motions of the peptide between the two binding modes (blue: mode 1, red: mode 2, pink: Pro4 to Pro7 in mode 1, yellow: Pro4 to Pro7 in mode 2). For Pro4 to Pro7 a rotation is the principle component of motion, while for other residues in the peptide a translation is the principle component of motion. Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 29 register-shift hypothesis supported by experiments WT A C D E F G H I K L M N P Q R S T V W Y T V W Y S H R P P P P G H R V Figure 7 CD2BP2-GYF tested with G8W mutant WT A C S H R D E F G H I K L M N P Q R S WT A C D E F G H I K L M N P Q R S T V W Y S H P R P PP WP H RP VP W H R V P Substitution analysis of the SHRPPPPWHR peptide binding to the GYF domain. All single substitution analogues of the peptide were synthesized on a cellulose membrane. The single letter code above each column marks the amino acid that replaces the corresponding wild-type residue, while the row defines the position of the substitution within the peptide. Spots in the most left column (WT) have identical sequences and represent the wild type peptide. The membrane was incubated with a GST-GYF construct of CD2BP2. Bound protein was detected with an anti-GST primary antibody and a horse-radish peroxidase coupled secondary antibody. The relative spot intensities correlate qualitatively with the binding affinities Gu et al. Biochemistry, in press (2005) 4. Lecture SS 20005 Cell Simulations 30 Summary - Complexes of adaptor domains with proline rich sequences form an important cellular network - Specificity of interactions vs. Multiplicity of interactions. - Interactions can be influenced by proline conformation (cis/trans) - Binding modes may not correspond to simple rigid body docking (see register shift) - Next 2 lectures of this module: set up and analyze interaction network with Cytoscape 4. Lecture SS 20005 Cell Simulations 31