Molecular Recognition Laboratorium

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Contact Information

Molecular Recognition Laboratorium,
Institute für medizinische Immunologie
CHARITÉ - UNIVERSITÄTSMEDIZIN BERLIN

	Hessische Str. 3-4
	D-10115 Berlin, Germany
	
	phone  +49-30-450 524092 
	fax    +49-30-450 524942 
	mail   annette.hayungs@charite.de
 	web    charite.de



Contents

Group Leader

Rudolf Volkmer

	Tel.:	+49 30 450 524 267
	Fax:	+49 30 450 524 942
	E-Mail:	rve(at)charite.de
	
	



Group Members

  • Bernhard Aÿ, Postdoc
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	bernhard.giese(at)charite.de
	
	
  • Prisca Boisguérin, Postdoc
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	prisca.boisguerin(at)charite.de
	
	
  • Zerrin Fidan, Doktorandin
	Tel.:	+49 30 450 524 285
	Fax:	+49 30 450 524 942
	E-Mail:	zerrin.fidan(at)charite.de
	
	
  • Annette Hayungs - Secretary
	Tel.:	+49 30 450 524 092
	Fax:	+49 30 450 524 942
	E-Mail:	annette.hayungs(at)charite.de
	
	
  • Marc Hovestädt - Doktorand
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	marc.hovestaedt(at)charite.de
	
	
  • Ines Kretzschmar - CTA
	Tel.:	+49 30 450 524 253
	Fax:	+49 30 450 524 942
	E-Mail:	ines.kretzschmar(at)charite.de
	
	
  • Christiane Landgraf - CTA
	Tel.:	+49 30 450 524 253
	Fax:	+49 30 450 524 942
	E-Mail:	chl(at)charite.de
	
	
	Tel.:	+49 30 450 524 278
	Fax:	+49 30 450 524 942
	E-Mail:	carsten.mahrenholz(at)charite.de
      
      
      
      
      
      
      
      
      
  • Judith Müller - Doktorandin
	Tel.:	+49 30 450 524 046
	Fax:	+49 30 450 524 942
	E-Mail:	judith.mueller(at)charite.de
	
	
  • Livia Otte - Postdoc
	Tel.:	+49 30 450 524 146
	Fax:	+49 30 450 524 942
	E-Mail:	livia.otte(at)charite.de
	
	
  • Rolf-Dietrich Stigler - IT- u. Sicherheitsbeauftragter
	Tel.:	+49 30 450 524 268
	Fax:	+49 30 450 524 942
	E-Mail:	rolf.stigler(at)charite.de
	
	
 	Tel.:	+49 30 450 524 046
	Fax:	+49 30 450 524 942
	E-Mail:	victor.tapia(at)charite.de
	
	
	
  • Julia Triebus - Diplomantin
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	julia.triebus(at)charite.de
	
	
  • Lars Vouillème - Doktorand
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	lars.vouilleme(at)charite.de
	
	
  • Eike Wolter - Diplomant
	Tel.:	+49 30 450 524 254
	Fax:	+49 30 450 524 942
	E-Mail:	julia.triebus(at)charite.de
	
	



Research interest

Technological Development of the Peptide Array Technologies

by Victor Tapia
FIGURE


A_ B_

PEPTIDE ARRAYS The combination of SPOT peptide synthesis (figure A, steps 1 to 4) with
appropriate immobilization techniques on glass supports (figure A, steps
5 and 6) is wide spread. The SPOT technology provides low-scale but
high-throughput synthesis, while immobilization of pre-synthesized
peptides offers the benefit of a "chemical" purification step and
flexible array design. Additionally, the glass support is compatible
with fluorescence detection (see figure B, adapted from the web) and
offers the possibility to miniaturize binding assays. Beyond economy,
the later point is essential for quantitative measurements at the
steady-state of binding activity, as has been described [Ekins 1998] and
can be proven by the mass-action law.



The basic point of this technology is the simultaneous display of a
systematic collection of peptides on a planar support, on which numerous
bimolecular interaction assays can be carried out under homogeneous
conditions.


Structural Modularity in Protein-Protein Recognition

by Victor Tapia
FIGURE


text

Protein Interaction Domains

The organisation of living systems is a complex network of molecular
interactions. Proteins are a central component of the network as they
may bind to other proteins as well as to phospholipids, nucleic acids
and small molecules to interconnect the diverse physiological functions
of the cell. In the background of these observations, the existence of a
molecular recognition code for cellular organisation is very suggestive.
MORE TEXT


Structural analysis of functional protein complexes suggests at least
two classes of protein-protein interaction that may be extendable to the
other kinds of protein interactions. In the first class, the
complementary surfaces of the interacting partners are both extensive.
Under these circumstances, the residues involved in each interacting
surface come together only upon protein folding. The second class
consists on asymmetric interactions, where a protein domain (folding
motive of moderate size like a pocket on the protein’s surface) may dock
a short lineal peptide motive (a peptide ligand) on the partner protein.
While interactions over extensive surfaces cannot be inferred, the
binding determinants of a protein interaction domain (PID) may be mapped
to short peptides matching the sequence of the ligand peptide. The
importance of small recognition domains in the formation of protein
complexes by binding to short lineal peptides was demonstrated in the
late 1980s and early 1990s (Sadowski, I. et al., 1986; Ren, R. et al.,
1993; Mayer, B.J. et al., 1993).

In this era of extensive
genome sequencing, many PIDs have been discovered. The interaction
partners and, therefore, the functions of such proteins may be
determined by identifying the critical binding sites for one family
member through evolutionary tracing (Lichtarge, O. et al., 1996) or
through high-parallel screening of functional protein arrays (Phizicky,
E. et al, 2003). Many of the PIDs in proteins can be grouped into
families that show clear evidence of their evolution from a common
ancestor, and genome sequences from Saccharomyces cerevisiae to Homo
sapiens reveal large numbers of proteins that contain one or more common
domains.


Src Homology Families - A PID Prototype

In a pioneering work on the kinase function and transforming activity of
the Fujinami Sarcoma Virus, Sadowski et al. (1986) discovered “a unique
domain… (which) is absent from kinases that span the plasma membrane”
and concluded that “the presence of this noncatalytic domain in all
known cytoplasmic tyrosine kinases of higher and lower eucaryotes argues
for an important biological function... the noncatalytic domain may
direct specific interactions of the enzymatic region with cellular
components that regulate or mediate tyrosine kinase function”. These
regions were called Src homology 2 (SH2) and 3 (SH3), the name SH1 being
reserved to the catalytic region. Since then, the gained knowledge on SH
domain function has been a paradigm in our understanding of PID
biochemistry.
MORE TEXT


The structure of SH2 family members involves about 100 residues that, in
the case of the kinase Src, are located N terminal to the catalytic
region and resembles a pocket dominated by a ß-sheet sandwiched between
a pair of a-helices. SH2 domains bind the protein containing them to a
second protein on a phosphorylated tyrosine residue (pY) in a specific
amino acid sequence context (Ladbury, J.E. et al., 2000).

The SH3 domain structure, also found in cytoplasmic kinases like Src,
consists largely of two ? sheets that form a partly open ß-barrel. The
ligand-binding site is a hydrophobic surface showing three shallow
pockets or grooves defined by conserved aromatic residues. The ligand
adopts an extended, left-handed helical conformation termed the
polyproline-2 (or PPII) helix. Two of the binding pockets of the SH3
domain are occupied by two hydrophobic proline dipeptides on two
adjacent turns of the helix, whereas the third ‘specificity’ pocket in
most cases interacts with a basic residue in the ligand distal to the
xPxxP core conserved motive of the PPII helix (Mayer, B.J. et al.,
2001).

The amino acids located at the binding site for the
phosphorylated polypeptide of SH2 and for the polyprolin core of SH3
have been the slowest to change during the long evolutionary process
that produced the large SH2 and SH3 families of peptide recognition
domains. Because mutation is a random process, this result is attributed
to the preferential elimination during evolution of all organisms whose
SH domains became altered in a way that inactivated the SH-binding site,
thereby destroying the function of the SH domain. Are the PID/ligand
interactions specific enough or must a certain interaction compete with
the bulk of structurally similar structures in a struggle for dynamical
complex formation?


From Promiscuous Recognition Events to Mutually Exclusive Cellular Responses

UPCOMMING: list of cited authors

The elucidation of functional pathways of signal transduction,
biochemical function or gene regulation, is firstly addressed in
proteomics by deriving interaction networks depicting ideally all
interactions in the cell. Several attempts have been done in this
direction on different model organisms and with varied methods,
including co-purification by affinity chromatography (Ho, Y. et al.,
2002; Gavin, A.-C. et al., 2002; Bouwmeester, T. et al., 2004), yeast
two-hybrid, phage display, peptide array technologies, etc. A comparison
of datasets derived by individual methods demonstrates that
different methods have different potential. For example, affinity
chromatographic approaches are biased to tight interactions such as
those involving extensive complementary surfaces, while
interactions in which one of the two partners contains at least one
PID are more frequent in the two-hybrid database. The higher
sensitivity of the so called synthetic approaches (yeast
two-hybrid, phage display and peptide array technologies) make them
better suited for detecting PID-mediated interactions since their
peptide affinity in terms of Kd falls in the 10 – 100 µM range (high Kd
--> low affinity). However, this advantage is counterbalanced by a low
specificity, especially of the yeast two-hybrid approach (Phizyki, 2002).
MORE TEXT


In order to correct this deficiency a double check-up of the information
fed into the interaction databases is recommended. This can be achieved
by deriving two interaction networks through orthogonal (fundamentally
different) synthetic methods and then considering only the intersection
between the two datasets (Tong, A.H.Y. et al.,2002; Castagnoli, L. et
al., 2004; Landgraf, C. et al., 2004). False positive reports are thus
reduced if the causes for measurement error are different in each
method.

The strength of this combined approach to deliver
physiologically relevant interactions has been proven for a phage
display/yeast two-hybrid intersected dataset (Tong, A.H.Y. et al.,2002).
A notable conclusion of this approach is that the intersected dataset of
proteins that are able to interact with a given PID is larger than
expected when cellular events are viewed as a precise wiring of the
proteins in the cell. Although a set of these biochemically potential
binders may have no physiological relevance due to expression at
different times or tissues, in vitro disrupted structures, etc., the
paradox of promiscuous recognition and mutually exclusive responses
seems to be inherent to PID mediated interactions: the recent work of
Landgraf et al. (2004) supports the observation that a large fraction of
natural peptides with the biochemical potential to bind to any given SH3
domain is actually used in vivo to mediate the formation of a
complex.

An additional difficulty to derive functional
interaction pathways is that the difference in affinity between
‘specific’ and ‘non-specific’ interactions has been shown to be less
than two orders of magnitude in the case of SH2 and its peptide ligands
(Songyang, Z. et al., 2004). Even when granted that the recognition
specificity of intact proteins by SH3 domains is greater than for
SH3-peptide recognition, affinity is not raised above one order of
magnitude (Arold, S. et al., 1998; Lee, C.H. et al., 1995). Moreover,
the ability of a point-mutant Src SH2 domain to effectively substitute
for the SH2 domain of the Sem-5 protein in activation of the Ras pathway
in vivo emphasises that the specificity of Sh2-mediated interactions is
not great (Marengere, L.E. et al., 1994). Consider the later statements
under the light of the fact that the affinity of the protein OppA for
its ligands is in the range of two orders of magnitude (Oppa is involved
in the mopping of peptides in the bacterial periplasm exhibiting no
sequence specificity). Tu put it all into a nutshell: the described
facts lead to a view of large and promiscuous SH-mediated interaction
networks.

Since it is possible to generate mutant SH3 domains
that have up to 40-fold higher affinity than their wild-types (Hiipakka
et al, 1999) , the potential of these domains as research tools and as
source of lead compounds for pharmaceutical development can not be
overseen. Furthermore, a question cannot be overheard in our minds:
which is the functional advantage of maintaining relative low affinity
and selectivity for PID-mediated interactions, instead of optimizing the
potential affinity of PIDs? And further: how can PID-dependent
interaction pathways achieve precise cellular responses?
A comfortable view is that sufficient effective selectivity can be brought
by compartmentalization, additive effects of multiple separate
interactions, cooperative assembly of multiprotein complexes, etc. and
that this effects can sustain linear functional pathways. An interesting
insight into this mater has been advanced by Zarrinpar, A. et al. In
their work (2003) the authors find out that while metazoan SH3 domains
may rescue the functionality of mutated Sho1-SH3 of the yeast, in the
set of yeast-own SH3 domains this promiscuity is forbidden. They thus
conclude and confirm that, on the background of diverging SH3 domains,
negative selection has drifted the ligand sequences to non-overlapping
areas of the particular SH3 binding regions on the sequence space
(please see Figure 1 and section 3.2 for further explanation of the
concept). Alternatively, a divergence process of the shape of the
binding region may be ‘guided’ by positive selection to avoid
overlapping and, thus, promiscuitive interactions. Nevertheless, the
picture of linear functional pathways is being revolutionized by a more
probabilistic view of a dynamical equilibrium between multiple
interactions, in which “the central organizing principle is a vast and
ever-shifting web of interactions, from which output is gauged by global
changes in complex binding equilibria” (Mayer, B. J., 2001).


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