(Received for publication, August 10, 1995; and in revised form, September 7, 1995)
From the
The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins.
Src homology (SH) The proposed
consolidated ligands are similar in concept to affinity reagents, with
the modification that the second functionality is a binding element
rather than a reactive moiety. Consolidated ligands may also be useful
reagents for studies of the cell biology of the signal transduction
complexes (for example, in additional combination with antibodies or
reporter groups like fluorescent tags), and may provide leads into
possible classes of diagnostic and therapeutic agents in the many areas
of pathology in which SH domains are involved. This approach will be
useful where micromolar affinities of ligands to single SH domains
provide insufficient affinity, and hence specificity, for
pharmacological action (e.g.(7) and (8) ). The protein target for this study is the regulatory apparatus,
SH(32) (4) , of human Abelson kinase (Abl). Abl was originally
isolated as the gene product from abl of murine leukemia
virus; the human abl gene has been isolated and shown, in
cases of chronic myologenous leukemia, to be a causative factor in a
fusion following chromosomal translocation (reviewed in (9) ).
Ligands of moderate affinity have been identified for the isolated SH2 (10) and SH3 domains (11, 12) . For Abl SH3, a
crystal structure of the complex of the ligand 3BP-1 is
available(13) , indicating that the ligand is in the so-called
class I orientation (reviewed in (2) ). Using the general
positioning of ligands to SH2 (1) as a framework, the likely
orientation of the 2BP-1 ligand, PVY*ENVamide(10) , was
modeled. In an SH(32) model (Fig. 1), these ligands are closest
at their C termini, and there is more than 20 Å separating them;
about 32 Å separate the C terminus of the SH3 ligand from the N
terminus of the SH2 ligand. An initial design of a consolidated ligand
for both domains uses this model.
Figure 1:
Model of the orientations of
SH2 and SH3 ligands in a consolidated ligand (I) on the Abl SH(32)
protein. Coordinates of individual SH2 and SH3 domains (6, 18) were fitted to the approximate orientation of
Lck SH(32) in the observed crystal structure(4) . The peptide
(I) was created and aligned to the expected binding positions based on
modelling of SH2 ligands to Src SH2 (20) and SH3 ligands to
Abl(16) . The positioning of glycyl and branched lysyl residues
is arbitrary, other than the restrictions of the subligand positioning.
Modeling and display used the Biosym system on an SGI
Iris.
Consolidated ligand I (Table 1) then joins the C termini of the individual 2BP and
3BP ligands using a branch through the side chain of a lysine residue.
By model building, the methylene segments of the lysyl side chain and
the oligoglycyl linker are expected to provide sufficient separation
between the C termini of the individual ligands. A series of
consolidated ligands, with different ligand segments for SH3, different
lengths and orientations of the linker, and different analogs of the
phosphotyrosyl residue in the ligand for SH2, were synthesized and
tested for affinity to Abl SH(32), and in some cases to SH3 and SH2.
This design and synthesis of consolidated ligands is shown to be
effective in Table 1. Increases in affinity of approximately 2
orders of magnitude were observed compared to unbranched equivalents (IversusII, IVversusV). An order of magnitude increase was observed comparing
the most strongly bound single ligand to the equivalent consolidated
ligand (2BP1versusIV). The affinity of a
consolidated ligand changed when a subligand was modified
proportionally to the change of affinity of separate ligand (I/3BP-1versusIV/3BP-2). The simple linker chemistry used
did not interfere with the subligands affinities to the individual SH3
or SH2 domains. Experiments with the single ligands as inhibitors show
classical competitive binding. The values of affinities of ligands with
variable linker lengths (III-G The affinities in Table 1might
be expected, from simple physical chemical concepts, to be somewhat
higher for the consolidated ligands. The thermodynamic treatment of the
consolidation of fragment affinities has been addressed by
Jencks(24) . Briefly, for affinities to SH2, SH3, and SH32, the
free energy contributions may be related by There are many possible
applications of these consolidated ligands and their analogs.
The examples in Table 1show that for Abl SH(32), the two ligand
sites can be oriented as shown in Fig. 1, that the 3BP-1 and
3BP-2 ligands for SH3 (in I and IV) are bound in the same
direction, and that the SH2 and SH3 binding sites do not interfere with
each other. Applications are expected to other SH(32)-containing
proteins for the purpose of similar mapping, or of obtaining high
affinity and specificity of binding for investigational or therapeutic
purposes(14) . Some examples are dual and higher subligand
consolidations for the adaptor protein Grb-2, and reagents like those
shown here for Abl, although possibly linear rather than
branched(15) , for Src family tyrosyl kinases(16) . A
major challenge in the investigation of intracellular signal
transduction involves the relatively transient nature of the signal
transducing complexes formed. Highly specific reagents may permit
trapping of, or selective interference with, these complexes. The
subligands in such consolidated ligands may be extended to other
components of signal transducing complexes, for example the active
sites of kinases, or binding sites of pleckstrin homology domains. For
Abl, the consolidated ligands shown here are possible early leads to
more complex ligands, which may have sufficient affinity and
specificity to block the Bcr-Abl fusion kinase that is the predominant
pathogenic agent in chronic myologenous leukemia. The chemistry used in
these consolidated ligands is relatively simple and was selected to
provide flexible linkers, as has been done for the
hirulogs(17) . Linkers of the correct size and rigidity should
provide greater affinities. The structure of the Abl
SH(32)/consolidated ligand complex may permit rational design of
linkers of an optimal rigidity and size, and also permit identification
of additional interactions for increased affinity. Peptidomimetic and
non-peptidic linkers are obviously practical, as are other subligands
including lipids, steroids, carbohydrates, and nucleic acids. These
consolidated ligands present novel opportunities for chemistry at the
interface with biology.
domains are building blocks in
many proteins involved in intracellular signal transduction. Detailed
understanding of the pathways involving these domains is complicated by
the substantial range of individual specificities in SH2 and SH3
domains, and their combination into large proteins that can form
multiple homo- and heteromolecular associations. The reductionist
approach of studying individual domains has been very
successful(1, 2, 3) . Nonetheless, the
interactions between the domains are still poorly understood. These
interactions are likely to be of significance in explaining more fully
the complete activities of the signal-transducing complexes. A detailed
structural picture of the inter- and intramolecular organization of the
domains is then a significant objective. Several cases of multiple SH2
and SH3 domain-containing constructs have been studied structurally
(Lck SH(32) ((4) ), Grb2 SH(323) ((5) ), and Abl SH(32) ((6) )), but there are technical limitations to current
structural approaches. In the crystalline state, packing forces may be
of the same magnitude as the weak interdomain forces, so there are
limits to the interpretation of diffraction studies. Solution studies
by NMR are only applicable in a molecular mass range less than
30
kDa. For NMR, time-averaged NOE constraints in rapidly exchanging
conformations are not readily interpretable. As a complement to direct
structural methods, we propose the investigation of such multidomain
complexes using ``consolidated'' ligands. These ligands,
having multiple binding portions, may be expected to bind with high
affinity and specificity when a linker between the two affine segments
is of the correct length, and there is little affinity of the linker
itself. The consolidated ligands do not necessarily resemble any
natural ligand. Such ligands are demonstrated here, binding to the
SH(32) of Abelson kinase. It is reasonable to assume that consolidated
ligands for dual SH2, dual SH3, or multiple combinations with other
ligands can be produced using a similar approach.
, IV-G
, VI-G
, VII-G
) are most readily interpreted as indicating an
optimal length of about G
, with little interaction between
the linker and the SH(32) protein. Analogs of the 2BP ligand of IV, namely the H-phosphonate (VIII), and the pY
Y of IV (IX) lead to decreases in affinity. The simplestmodel
of how the consolidated ligand binds is that there are specific
contacts at the subligand sites, and a solvated linker region. Using a
model derived from NMR studies of the SH3 and SH2 domains(6) ,
assuming that the orientation for Abl is similar to that seen in
Lck(4) , the two subligands are correctly accommodated, and no
specific interactions are forced on the linker segment (Fig. 1).
The specificity of interaction is much increased also (Table 1),
so that IV will now discriminate between the complete SH(32) and
its subdomains by more than an order of magnitude. The decreased
affinity of IV compared to 2BP1 for the single SH2 domain is not
readily explained. One possibility is that IV forms a partially
folded structure reducing the population of ligand in a conformation
suitable for binding to SH2.
G°
=
G
+
G
+
G
where the subscripts 32, 2, and 3 refer to the SH domain
interaction, and the superscript i refers to the individual binding contributions, and
G
represents
the change in probability of binding that results from the connection
of the two ligands. It may be assumed that the principal method by
which apparent affinity increases for the ligands of this work is the
reduction in degrees of translational freedom, when one subligand is
bound(24) . For perfect linker length and geometry, a
consolidated ligand affinity up to the product of the individual
association constants might be expected (i.e.
G°
=
G
+
G
). Increases in degrees of
rotational freedom associated with each glycyl residue, and with the
lysyl methylenes reduce this affinity considerably, that is
G
represents a large, energetically
unfavorable, increase in entropic contribution, and the overall free
energy of interaction of the consolidated ligand is enhanced, but not
dramatically, over that of its components.