V3 From Protein Complexes to Networks and back

1
V3 From Protein Complexes to Networks and back

• Protein networks could be defined in a number of
  ways
• (1) Co-regulated expression of genes/proteins
• (2) Proteins participating in the same metabolic
  pathways
• (3) Proteins sharing substrates
• (4) Proteins that are co-localized
• (5) Proteins that form permanent supracomplexes
  protein machines
• (6) Proteins that bind each other transiently
• (signal transduction, bioenergetics ... )
• In V4 we will look at computational methods to
  predict protein-protein interactions.
• Today, we will look at permanent and transient
  protein complexes.

2
Methods for the structural characterization of
macromolecular assemblies
(a) Electron diffraction map and 3D X-ray protein
structure. X-ray provides atomic-resolution
structures. (b) 3D protein structure and plot
showing chemical shifts determined by NMR. NMR
spectroscopy extracts distances between atoms by
measuring transitions between different nuclear
spin states within a magnetic field. These
distances are then used as restraints to build 3D
structures. NMR spectroscopy also provides
atomic-resolution structures, but is generally
limited to proteins of about 300 residues. It
plays an increasingly important role in studying
interaction interfaces between structures
determined independently. (c) EM micrograph and
3D reconstruction of a virus capsid. EM is based
on the analysis of images of stained particles.
Different views and conformations of the
complexes are trapped and thus thousands of
images have to be averaged to reconstruct the
three-dimensional structure. Classical
implementations were limited to a resolution of
20 Å. More recently, single-particle cryo
techniques, whereby samples are fast frozen
before study, have reached resolutions as high as
approximately 6 Å. EM provides information about
the overall shape and symmetry of macromolecules.
(d) Slice images and rendered surface of a
ribosome-decorated portion of endoplasmic
reticulum. In electron tomography, the specimen
studied is progressively tilted upon an axis
perpendicular to the electron beam. A set of
projection images is then recorded and used to
build a 3D model. This technique can tackle large
organelles or even complete cells without
perturbing their physiological environment. It
provides shape information at resolutions of
approximately 30 Å. (e) Yeast two-hybrid array
screen and small network of interacting proteins.
Interaction discovery comprises many different
methods whose objective is to determine spatial
proximity between proteins. These include
techniques such as the two-hybrid system,
affinity purification, FRET, chemical
cross-linking, footprinting and protein arrays.
These methods provide very limited structural
information and no molecular details. Their
strength is that they often give a
quasi-comprehensive list of protein interactions
and the networks they form.
Russell et al. Curr. Opin. Struct. Biol. 14, 313
(2004)
3
Hybrid models docking X-ray structures into EM
maps
Hybrid assembly of the 80S ribosome from yeast.
(a) Superposition of a comparative protein
structure model (red) of a domain from ribosomal
protein L2 from Bacillus stearothermophilus with
the actual structure (blue) (PDB code 1RL2). (b)
A partial molecular model of the whole yeast
ribosome calculated by fitting atomic rRNA (not
shown) and comparative protein structure models
(ribbon representation) into the electron density
of the 80S ribosomal particle.
Russell et al. Curr. Opin. Struct. Biol. 14, 313
(2004)
4
Putative structure through modeling and
low-resolution EM
(a) Exosome subunits. The top of the panel shows
the domain organization of two subunits present
in the complex, but lacking any detectable
similarity to known 3D structures. The model for
the nine other subunits (bottom) was constructed
by predicting binary interactions using
InterPReTS and building models based on a
homologous complex structure using comparative
modeling. (b) EM density map (green mesh) with
the best fit of the model shown as a gray surface
and the predicted locations of the subunits
labeled. The question marks indicate those
subunits for which no structures could be modeled.
Russell et al. Curr. Opin. Struct. Biol. 14, 313
(2004)
5
Potential errors in biochemical interaction
discovery
(a) Indirect interactions between
cyclin-dependent kinase regulatory subunit (CKS)
and cyclin A detected by the Y2H system. Several
interactions between CKS domains and cyclins were
reported in genome-scale two-hybrid studies.
However, analysis of 3D structures suggests that
the endogenous cyclin-dependent kinase 2 (CDK2)
probably mediates the interaction, as combining
the CDK2CKS and CDK2cyclin A structures places
the CKS and cyclin domains 18 Å apart.
(b) An example of an interaction that is not
detected by any screen, possibly because
molecular labels (e.g. affinity purification
tags, or two-hybrid DNA binding or activation
domains) are interfering with the interaction.
The X-ray structure of the actinprofilin complex
reveals that the actin C terminus (C-t) lies at
the interaction interface (the other N and C
termini are also labeled).
Russell et al. Curr. Opin. Struct. Biol. 14, 313
(2004)
6
1 Protein-Protein Complexes
It has been realized for quite some time that
cells dont work by random diffusion of
proteins, but require a delicate structural
organization into large protein complexes. Which
complexes do we know?
7
RNA Polymerase II
RNA polymerase II is the central enzyme of gene
expression and synthesizes all messenger RNA in
eukaryotes.
Cramer et al., Science 288, 640 (2000)
8
RNA processing splicesome
Structure of a cellular editor that "cuts and
pastes" the first draft of RNA straight after it
is formed from its DNA template. It has two
distinct, unequal halves surrounding a tunnel.
Larger part appears to contain proteins and the
short segments of RNA, smaller half is made up
of proteins alone. On one side, the tunnel opens
up into a cavity, which is believed to function
as a holding space for the fragile RNA waiting to
be processed in the tunnel. Profs. Ruth and
Joseph Sperling http//www.weizmann.ac.il/
9
Protein synthesis ribosome
The ribosome is a complex subcellular particle
composed of protein and RNA. It is the site of
protein synthesis, http//www.millerandlevine.co
m/chapter/12/cryo-em.html
Model of a ribosome with a newly manufactured
protein (multicolored beads) exiting on the
right.
10
Signal recognition particle
Cotranslational translocation of proteins across
or into membranes is a vital process in all
kingdoms of life. It requires that the
translating ribosome be targeted to the membrane
by the signal recognition particle (SRP), an
evolutionarily conserved ribonucleoprotein
particle. SRP recognizes signal sequences of
nascent protein chains emerging from the
ribosome. Subsequent binding of SRP leads to a
pause in peptide elongation and to the ribosome
docking to the membrane-bound SRP receptor. SRP
shows 3 main activities in the process of
cotranslational targeting first, it binds to
signal sequences emerging from the translating
ribosome second, it pauses peptide elongation
and third, it promotes protein translocation by
docking to the membrane-bound SRP receptor and
transferring the ribosome nascent chain complex
(RNC) to the protein-conducting channel.
40S small ribosomal subunit (yellow) 60S large
ribosomal subunit (blue), P-site tRNA (green),
SRP (red). Halic et al. Nature 427, 808 (2004)
11
Nuclear Pore Complex
A three-dimensional image of the nuclear pore
complex (NPC), revealed by electron microscopy.
A-B The NPC in yeast. Figure A shows the NPC
seen from the cytoplasm while figure B displays a
side view. C-D The NPC in vertebrate (Xenopus).
http//www.nobel.se/medicine/educational/dna/a/t
ransport/ncp_em1.html Three-Dimensional
Architecture of the Isolated Yeast Nuclear Pore
Complex Functional and Evolutionary
Implications, Qing Yang, Michael P. Rout and
Christopher W. Akey. Molecular Cell, 1223-234,
1998
NPC is a 50-100 MDa protein assembly that
regulates and controls trafficking of
macromolecules through the nuclear envelope.
12
GroEL a chaperone to assist misfolded proteins
Schematic Diagram of GroEL Functional States (a)
Nonnative polypeptide substrate (wavy black line)
binds to an open GroEL ring. (b) ATP binding to
GroEL alters its conformation, weakens the
binding of substrate, and permits the binding of
GroES to the ATP-bound ring. (c) The substrate
is released from its binding sites and trapped
inside the cavity formed by GroES binding. (d)
Following encapsulation, the substrate folds in
the cavity and ATP is hydrolysed. (e) After
hydrolysis in the upper, GroES-bound ring, ATP
and a second nonnative polypeptide bind to the
lower ring, discharging ligands from the upper
ring and initiating new GroES binding to the
lower ring (f) to form a new folding active
complex on the lower ring and complete the cycle.
http//people.cryst.bbk.ac.uk/ubcg16z/chaperone
.html
Ransom et al., Cell 107, 869 (2001)
13
Arp2/3 complex
The seven-subunit Arp2/3 complex choreographs the
formation of branched actin networks at the
leading edge of migrating cells. (A) Model of
actin filament branches mediated by Acanthamoeba
Arp2/3 complex. (D) Density representations of
the models of actin-bound (green) and the free,
WA-activated (as shown in Fig. 1D, gray) Arp2/3
complex.
Volkmann et al., Science 293, 2456 (2001)
14
proteasome
The proteasome is the central enzyme of
non-lysosomal protein degradation. It is involved
in the degradation of misfolded proteins as well
as in the degradation and processing of short
lived regulatory proteins.The 20S Proteasome
degrades completely unfoleded proteins into
peptides with a narrow length distribution of 7
to 13 amino acids. http//www.biochem.mpg.de/xray
/projects/hubome/images/rpr.gif Löwe, J., Stock,
D., Jap, B., Zwickl, P., Baumeister, W. and
Huber, R. (1995). Crystal structure of the 20S
proteasome from the archaeon T. acidophilum at
3.4 Å resolution. Science 268, 533-539.
15
Energy conversion Photosynthetic Unit
Structure suggested by force field
based molecular docking. http//www.ks.uiuc.edu/R
esearch/vmd/gallery
16
icosahedral pyruvate dehydrogenase complex a
multifunctional catalytic machine
Model for active-site coupling in the E1E2
complex. 3 E1 tetramers (purple) are shown
located above the corresponding trimer of E2
catalytic domains in the icosahedral core. Three
full-length E2 molecules are shown, colored red,
green and yellow. The lipoyl domain of each E2
molecule shuttles between the active sites of E1
and those of E2. The lipoyl domain of the red E2
is shown attached to an E1 active site. The
yellow and green lipoyl domains of the other E2
molecules are shown in intermediate positions in
the annular region between the core and the outer
E1 layer. Selected E1 and E2 active sites are
shown as white ovals, although the lipoyl domain
can reach additional sites in the complex.
Milne et al., EMBO J. 21, 5587 (2002)
17
Apoptosome
(A) Top view of the apoptosome along the 7-fold
symmetry axis. (B) Details of the spoke. (C) A
side view of the apoptosome reveals the unusual
axial ratio of this particle. The scale bar is
100 Å. (D) An oblique bottom view shows the
puckered shape of the particle. The arms are bent
at an elbow (see asterisk) located proximal to
the hub. Acehan et al. Mol. Cell 9, 423 (2002)
Apoptosis is the dominant form of programmed cell
death during embryonic development and normal
tissue turnover. In addition, apoptosis is
upregulated in diseases such as AIDS, and
neurodegenerative disorders, while it is
downregulated in certain cancers. In apoptosis,
death signals are transduced by biochemical
pathways to activate caspases, a group of
proteases that utilize cysteine at their active
sites to cleave specific proteins at aspartate
residues. The proteolysis of these critical
proteins then initiates cellular events that
include chromatin degradation into nucleosomes
and organelle destruction. These steps prepare
apoptotic cells for phagocytosis and result in
the efficient recycling of biochemical
resources. In many cases, apoptotic signals are
transmitted to mitochondria, which act as
integrators of cell death because both effector
and regulatory molecules converge at this
organelle. Apoptosis mediated by mitochondria
requires the release of cytochrome c into the
cytosol through a process that may involve the
formation of specific pores or rupture of the
outer membrane. Cytochrome c binds to Apaf-1 and
in the presence of dATP/ATP promotes assembly of
the apoptosome. This large protein complex then
binds and activates procaspase-9.
18
Future?

• Structural genomics (X-ray) may soon generate
  enough templates of individal folds.
• Structural genomics may be expanded to protein
  complexes.
• Interactions between proteins of the same fold
  tend to be similar when the sequence identity is
  above approximately 30 (Aloy et al.).
• Hybrid modelling of X-ray/EM will not be able to
  answer all questions
• problem of induced fit
• transient complexes cannot be addressed by these
  techniques
• ? Essential to combine large variety of hybrid
  complementary methods
• Russell et al. Curr. Opin. Struct. Biol. 14, 313
  (2004)

19
2 Information on protein-protein networks
20
2. Yeast 2-Hybrid Screen
Data on protein-protein interactions from Yeast
2-Hybrid Screen. One role of bioinformatics is
to sort the data.
21
Protein cluster in yeast
Cluster-algorithm generates one large cluster
for proteins interacting with each other based
on binding data of yeast proteins.
Schwikowski, Uetz, Fields, Nature Biotech. 18,
1257 (2001)
22
Annotation of function
After functional annotation connect clusters
of interacting proteins.
Schwikowski, Uetz, Fields, Nature Biotech. 18,
1257 (2001)
23
Annotation of localization
Schwikowski, Uetz, Fields, Nature Biotech. 18,
1257 (2001)
24
3 Systematic identification of protein complexes
25
Systematic identication of large protein complexes
Yeast 2-Hybrid-method can only identify binary
complexes. Cellzome company attach additional
protein P to particular protein Pi , P binds to
matrix of purification column. ? yields Pi and
proteins Pk bound to Pi .
Identify proteins by mass spectro- metry
(MALDI- TOF).
Gavin et al. Nature 415, 141 (2002)
26
Analyis of protein complexes in yeast (S.
cerevisae)
Identify proteins by scanning yeast
protein database for protein composed of
fragments of suitable mass. Here, the
identified proteins are listed according to
their localization (a). (b) lists the number
of proteins per complex.
Gavin et al. Nature 415, 141 (2002)
27
Validation of methodology
Check of the method can the same complex be
obtained for different choice of attachment
point (tag protein attached to different
coponents of complex)? Yes (see gel).
Method allows to identify components of complex,
not the binding interfaces. Better for
identification of interfaces Yeast 2-hybrid
screen (binary interactions). 3D models of
complexes are important to develop inhibitors.

• theoretical methods (docking)
• electron tomography

Gavin et al. Nature 415, 141 (2002)
28
Analysis of affinity-purified protein complexes
in E.coli
TAP-purification for 25 of the E.coli genome,
targeting 1000 ORFs. ? 857 tagged proteins,
including 198 essential and conserved proteins ?
648 could be purified. Out of these, 118 had no
detectable partners. 530 other baits 5254
protein-protein interactions. Verification by
reciprocal tagging of many candidate partners
53 validation rate (716 non-redundant validated
interactions). 85 of the validated interactions
are new! They were not contained sofar in the
Database of Interacting Proteins (DIP),
Biomolecular Interaction Network Database (BIND),
and other databases.
Butland et al. Nature 433, 531 (2005)
29
Pilot purification of DNA-dependent RNA polymerase
SDSPAGE silver-stain analysis of the components
of affinity-purified complexes from E. coli.
ac, Purification of TAP-tagged E. coli RNAP
subunit b (a) and two associated proteins
SPA-tagged b1731 (b) and TAP-tagged YacL (c).
a Tagged core subunit ? of RNA polymerase (RpoB)
co-purified specifically with essential
elongation factors (NusA and NusG), specified
sigma factors involved in promoter recognition
(RpoH, RpoS, RpoD) and with accessory factors
(RpoZ, HepA and YacL). Similarly, NusG was
co-purified with YacL, HepA, core enzyme and
termination factor Rho, whereas RpoZ bound RpoD,
NusA and b1731 (sofar unknown). b reciprocal
experiment tagged b1731 co-purified with ?,
RpoC, RpoA, RpoD and RpoZ, but not with Nus
factors, HepA or YacL. ? probably b1731
exclusively binds to ?, suggesting an exclusive
association with initiating holoenzyme. c
However, tagged YacL bound RpoZ, NusG and HepA
together with core enzymes, suggesting a role in
elongation.
Butland et al. Nature 433, 531 (2005)
30
Network properties of bacterial protein-protein
interactions
Network of validated protein complexes.
Interactions are represented as directional edges
extending from the tagged protein. Baits without
partners were removed for clarity. Red nodes,
essential proteins blue nodes, non-essential
proteins black ovals, complexes discussed in
text.
Butland et al. Nature 433, 531 (2005)
31
Significance of novel interactions? Check
functional annotation
E.g., acyl carrier protein (ACP), a key carrier
of growing fatty acid chains, bound specifically
and reproducibly to enzymes linked to biogenesis
of fatty acids, phospholipids and lipid A
(essential outer-membrane constituent), including
- two 3-ketoacyl-ACP synthases (FabB, FabF) -
3-ketoacyl-ACP reductase (FabG), -
3-hydroxyacyl-ACP dehydrase (FabZ), - LpxD
(essential protein required for lipid A
biogenesis), - YbgC (tol-pal cluster hydrolase of
short-chain acyl-CoA thioesters), - AcpS
(involved in transfer of 4 - phosphopantethein
to ACP), - Aas and PlsB (membrane proteins
involved in phospholipid acylation), and - YiiD
(putative acetyltransferase). ACP also
co-purified with GlmU (an essential
bi-functional enzyme that converts
glucosamine-1-phosphate to UDP-GlcNAc (lipid A
precursor)), AidB (isovaleryl-CoA dehydrogenase),
SecA (pre-protein translocase), as well as MukB
and SpoT.
Butland et al. Nature 433, 531 (2005)
32
Network properties of bacterial protein-protein
interactions
Connectivity distribution of validated
interactions k per protein plotted as a function
of frequency, p(k). Inset log-plot power law
distribution, p(k) lt k-?.
? evidence of scale-free behavior Comparable
connectivity observed for the essential-conserved
proteins alone.
Butland et al. Nature 433, 531 (2005)
33
Interaction network connectivity and robustness
Node shading (white to black) is scaled according
to the increasing number of genomes in which a
putative interaction is detected based on gene
co-occurrence. a, Interaction network after
attacking the 20 most highly connected, highly
conserved (detected by BLAST in gt 125 genomes)
hubs. b, Network before attack (See Fig 3c). ?
removal of 20 hubs markedly reduced network
connectivity.
Butland et al. Nature 433, 531 (2005)
34
Interaction network connectivity and robustness
c Connectivity properties of the conserved (blue
color detected by BLAST in ? 125 genomes) and
non conserved (purple color ? 25 genomes)
proteins. x-axis number of connections per
protein y-axis frequency of proteins belonging
to this group. Inset mean of random sets of
interacting proteins (Control) of the same size
as the datasets. d, x-axis number of
interactions per protein y-axis number of
genomes a homolog was detected in (BLAST score
50).
Hubs are all conserved!
Protein connectivity is proportional to the
number of homologous in other genomes
Butland et al. Nature 433, 531 (2005)
35
Network properties of bacterial protein-protein
interactions
Network of highly conserved proteins co-occurring
in 125 genomes (homologue raw BLAST bit score
50). The most highly conserved proteins are
highly connected, forming a single interconnected
component. This core set of interactions
(including the ribosome) potentially fulfils
critical roles across all bacteria.
Butland et al. Nature 433, 531 (2005)
36
Bioinformatic analyses of interacting protein
modules
a Node shading (white to black) is scaled
according to the increasing number of genomes in
which a putative interaction is detected based on
gene co-occurrence using COGs genomes. Similar
results as for highly conserved proteins.
b shows interaction network for proteins with
co-occurrence in 40 genomes. Proteins with no
interacting partner were removed for clarity.
Butland et al. Nature 433, 531 (2005)
37
4 Aim generate structures of protein complexes

• Experiment
• Start from 232 purified complexes from TAP
  strategy.
• Select 102 that gave samples most promising for
  EM from analysis of gels and protein
  concentrations.
• Take EM images.
• Theory
• Make list of components.
• Assign known structures of individual proteins.
• Assign templates of complexes
• If complex structure available for this pair
• if complex structure available for homologous
  protein
• if complex structure available for structurally
  similar protein (SCOP)

Bettina Böttcher (EM) Rob Russell (Bioinformatics)
38
How transferable are interactions?
analyze interaction similarity (iRMSD) vs.
sequence identity for all the available pairs of
interacting domains with known 3D
structure. Curve shows 80 percentile
(i.e. 80 of the data lies below the curve).
Points below the line (iRMSD 10 Å) are similar
in interaction.
Conclusion mode of interaction is conserved
among protein-protein complexes with gt 30 40
sequence identity.
Aloy et al. Science, 303, 2026 (2004)
39
Bioinformatics Strategy
Illustration of the methods and concepts used.
How predictions are made within complexes
(circles) and between them (cross-talk). Bottom
right shows two binary interactions combined into
a three-component model
Aloy et al. Science, 303, 2026 (2004)
40
3SOM algorithm vector-based circumference
superimposition
A 2D variant of the 3D vector-based surface
superimposition that is central to the 3SOM
algorithm. For each tested voxel a on the
circumference of the target, a vector va is
calculated that approximates the normal vector
orthogonal to the tangent line in a and with
origin in a. Vector va is superimposed on each
vector vb that is associated with a voxel b on
the circumference of the template. The
goodness-of-fit of the transformation in question
is assessed by measuring the circumference
overlap, the fraction of target circumference
voxels that is projected onto (or near) the
template circumference (triangles). In 3D, a
rotational degree of freedom is left around the
superimposed vectors, which is sampled in
rotational steps of 9.
Ceulemans, Russell J. Mol. Biol., 338, 783 (2004)
41
Successful models of yeast complexes
(A) Exosome model on PNPase fit into EM map. (B)
RNA polymerase II with RPB4 (green)/RPB7 (red)
built on Methanococcus jannaschii equivalents,
and SPT5/pol II (cyan) built with IF5A. (C and
D) Views of CCT (gold) and phosphoducin 2/VID27
(red) fit into EM map. (E) Micrograph of POP
complex, with particle types highlighted. (F)
Ski complex built by combination of two
complexes.
Aloy et al. Science, 303, 2026 (2004)
42
Cross talk between complexes
(Top) Triangles components with at least one
modelable structure and interaction squares,
structure only circles, others. Lines show
predicted interactions. Thick lines imply a
conserved interaction interface red, those
supported by experiment. (Bottom) Expanded view
of cross-talk between transcription complexes
built on by a combination of two complexes.
Aloy et al. Science, 303, 2026 (2004)
43
Summary
A combination of 3D structure and
protein-interaction data can already provide a
partial view of complex cellular structures.
The structure-based network derived from
cross-talk between complexes provides a more
realistic picture than those derived blindly from
interaction data, because it suggests molecular
details for how they are mediated. Of course,
the picture is still far from complete and there
are numerous new challenges. The
structure-based network derived here provides a
useful initial framework for further studies. Its
beauty is that the whole is greater than the sum
of its parts Each new structure can help to
understand multiple interactions. The complex
predictions and the associated network will thus
improve exponentially as the numbers of
structures and interactions increase, providing
an ever more complete molecular anatomy of the
cell.
Aloy et al. Science, 303, 2026 (2004)