4. Lecture SS 20005Cell Simulations1 V4: Area 3 – interaction networks Area 1:Systems of coupled...

4. Lecture SS 20005

Cell Simulations 1

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 2

SH3 as a structural motif in SRC tyrosine kinase

Zarrinpar, A., Bhattacharyya, R. P., and Lim, W. A. (2003) The structure and function of proline recognition domains, Sci. STKE. RE8.

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).

Domains that bind proline-rich motifs are critical to the assembly of many intracellular signaling complexes and pathways.

4. Lecture SS 20005

Cell Simulations 3

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 4

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.

4. Lecture SS 20005

Cell Simulations 5

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 SRC-

family kinases binds to the carboxy-

terminal 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.

Nature Reviews Immunology 1; 95-107 (2001)

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.

4. Lecture SS 20005

Cell Simulations 6

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 7

A proline-driven conformational switch within the Itk SH2 domain

Mallis, Brazin, Fulton, Andreotti, Structural characterization of a proline-driven conformational switch within the Itk SH2 domain, Nat. Struct. Biol. 9, 900 - 905 (2002)

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.

4. Lecture SS 20005

Cell Simulations 8

Structural differences between cis and trans isomers

Mallis, Brazin, Fulton, Andreotti, Structural characterization of a proline-driven conformational switch within the Itk SH2 domain, Nat. Struct. Biol. 9, 900 - 905 (2002)

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).

4. Lecture SS 20005

Cell Simulations 9

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.

4. Lecture SS 20005

Cell Simulations 10

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.

4. Lecture SS 20005

Cell Simulations 11

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.

4. Lecture SS 20005

Cell Simulations 12



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.

4. Lecture SS 20005

Cell Simulations 13



Another unique property of proline is that it is the only naturally occurring N-

substituted 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, sequence-

specific 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.

4. Lecture SS 20005

Cell Simulations 14



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.

4. Lecture SS 20005

Cell Simulations 15

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.

4. Lecture SS 20005

Cell Simulations 16

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.

4. Lecture SS 20005

Cell Simulations 17

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.

4. Lecture SS 20005

Cell Simulations 18

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

4. Lecture SS 20005

Cell Simulations 19

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.

4. Lecture SS 20005

Cell Simulations 20

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

4. Lecture SS 20005

Cell Simulations 21

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.

4. Lecture SS 20005

Cell Simulations 22

Identification of a novel “register-shifted” binding mode

Gu et al. Biochemistry, in press (2005)

NMR structure of GYF domain with wild-

type 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.

4. Lecture SS 20005

Cell Simulations 23

What is the conformation of the unbound peptide


4. Lecture SS 20005

Cell Simulations 24

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).

4. Lecture SS 20005

Cell Simulations 25

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.

4. Lecture SS 20005

Cell Simulations 26

Which residues are crucial for binding?


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

WT A C D E F G H I K L M N P R S T V W YQ

H

R

S

P

R

H

P

P

P

W

V


H

R

S

P

R

H

P

P

P

G

V

Conclusion:Two central prolines are criticaland the following glycine.But can this glycine be mutated to Trp?

4. Lecture SS 20005

Cell Simulations 27

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 CD2BP2-

GYF 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.

4. Lecture SS 20005

Cell Simulations 28

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.

4. Lecture SS 20005

Cell Simulations 29

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.

4. Lecture SS 20005

Cell Simulations 30

register-shift hypothesis supported by experiments


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


H

R

S

P

R

H

P

P

P

W

V


H

R

S

P

R

H

P

P

P

G

V

Figure 7

WT A C D E F G H I K L M N Q R S T WVP

HR

S

PRH

PP

PW

V

Y

CD2BP2-GYF tested with G8W mutant

4. Lecture SS 20005

Cell Simulations 31

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

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