Molecular matchmakers

DAGMAR RINGE

MACROMOLECULAR RECOGNITION

Molecular matchmakers Immunosuppressant drugs create new surfaces for interactions of their targets with other proteins, illustrating what may be a general principle of macromolecular recognition. There is a link missing between our understanding of individual proteins at the molecular level and of complex molecular systems at the cellular level. Recent discoveries about the mechanisms by which immunosuppressant drugs exert their effects have led to the realization that the 'missing link' is a special kind of molecular recognition. A set of molecules has been discovered that mediate interactions between different macromolecules. These matchmakers come in all sizes, from small molecules to proteins. They function by binding to one macromolecule and thereby creating a new recognition surface for interaction with a second macromolecule. I propose that these molecular matchmakers be called 'schatchens', a Yiddish word meaning marriage broker. Schatchens are the antithesis of chaperones. Chaperones were so named because they prevent undesirable associations. Schatchens promote desirable associations, required in processes such as signal transduction, transcription and antigen presentation. Although it might seem that the term 'schatchen' would incorporate allosteric effectors and any other molecule that, by binding to a protein changes its conformation so as to allow interactions with other molecules, I intend the definition to be more specific. By a schatchen, I mean a molecule that actually participates in the intermolecular association that it promotes. A schatchen is part of the binding surface, which is not generally true of allosteric regulators. I will summarize the general features of schatchens and how they mediate macromolecular interactions. Several exampies will be considered in detail, some of which are of considerable biomedical importance.

Double-sided sticky tape The concept of specific, mutual recognition by two proteins or by a protein and a ligand is a familiar one. Molecular recognition usually involves shape complementarity to provide specificity and numerous non covalent interactions to provide affinity [1]. Small changes in either shape or interaction complementarity very often lead to significant changes in affinity. Loss of a single hydrogen bond can lead to an almost ten-fold reduction in affinity, which can have drastic consequences for a cellular process [2]. From these considerations it is clear that interactions of moderate affinity do not require an extensive interaction surface. Macromolecular interactions of moderate affinity are very important in biology, as they are easily modulated. The problem for the cell, therefore, is to create small, specific sticky patches that can be removed when desired. The solution to this problem is a set of molecules that function like double-sided sticky tape. When such a molecule binds to a particular macromolecule - - which Volume 2

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it may do with very high affinity - - the complex presents a new surface that can be recognized by another macromolecule. The latter interaction may be of any affinity, but is usually less tight than the initial binding of the schatchen. When the matchmaker is small relative to the size of the macromolecule to which it first binds, it may function by inducing a conformational change in that molecule as well as by providing, a partial binding surface itself. The change often involves rearrangements to the conformations of surface loops, with the result that a new protein surface is presented. When the matchmaker is nearly equal in size to the protein with which it interacts, the new interacting surface is composed of nearly equal parts of the matchmaker and the macromolecule. In this case, the analogy to double-sided sticky tape is a precise one.

The immunophilins The fungal products FK506, cTclosporin A and rapamycin bind with high affinity to soluble, cytoplasmic receptor proteins, leading to inhibition of the transcription of a number of genes that are normally activated following stimulation of the T-cell receptor. This inhibition of Tcell activation is used clinically for the prevention of graft rejection after organ and bone marrow transplantation, hence the compounds are called immunosuppressants. The mechanism by which immunosuppressants function depends on the recognition of the drug-receptor complexes by other proteins in a cascade that forms part of a specific signal transduction pathway [3]. The proteins to which the immunosuppressants bind are called immunophilins: cyclophilin is the receptor for cyclosporin A, and FKBP (FK506-binding protein) the receptor for FK506 and rapamycin. Both of these proteins possess catalytic activity - - they catalyse the cis-trans isomerization of peptidyl-prolyl bonds [4,5]. For some time, it was thought that inhibition of this activity was responsible for the immunosuppressive effects of the drugs, isomerization of protein-containing peptide bonds being required for proper folding of certain proteins in the immune system. It now seems clear that this is not the case. Rather, FK506, cyclosporin A and rapamycin are schatchens: they act by forming a new surface, composed of parts of the immunophilin and parts of themselves. This new surface is selectively sticky for proteins important in T-cell activation. The proteins to which the drug receptor complexes bind are still being identified, but one has already been characterized in some detail [6]. Calcineurin, a calmodulin-dependent serine/threonine protein phosphatase, binds tightly to both FK506-FKBP and 545

cyclosporin A~zyclophilin complexes, but not to either the free drugs or free receptors. Binding of the complexes inhibits the phosphatase activity of calcineurin. Interestingly, the rapamycin-FKBP complex does not inhibit or even bind to calcineurin [3], so it must have another target. As rapamycin binds to FKBP at the same site as FK506, the implication is that the drugs themselves must form part of the binding surface, perhaps the large part, as the conformation of FKBP does not change much in the complex. Creation of this new binding surface appears to require a large conformational change - - not in the receptor but in the drug, in the case of cyclosporin A at least. In solution, this cyclic peptide has a conformation in which several of its hydrophobic side chains are directed inwards. NMR studies show that when bound to cyclophilin, however, cyclosporin A is almost literally turned inside-out (Fig. 1) [7,8]. In this conformation, some of the hydrophobic side chains of cyclosporin A are now able to interact with the immunophilin, whereas others are directed away from the receptor site, forming a patch on the surface of the immunophilin. Modelling studies of the cyclosporin A~yclophilin complex by a new dynamic technique [9] suggest that this new surface is very different in character from that presented by either molecule alone.

CyclosporinA ~

Cyclophilin

Calcineurin A

,@

bd Fig. 1. When cyclosporin A binds its cellular receptor, cyclophilin, it undergoes a dramatic conformational change so that it presents a surface complementary to the phosphatase calcineurin. As noted above, rapamycin is thought to work by the same mechanism, but with a different target. One such target has recently been identified - - the rapamycin-FKBP complex blocks the phosphorylation and activation of the 70 kD $6 protein kinases in a variety of animal cells ([10], and see the next article in this issue). FK506 has no effect on these kinases, either alone or in a complex with FKBP. Thus, although rapamycin and FK506 bind to identical sites on FKBP they create complementary sites for different partners. This illustrates the power of the schatchen mechanism

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for controlling cellular processes: by varying the matchmaker, associations can be formed between proteins that would not otherwise recognize each other, and these proteins can be present together without unwanted interactions until the schatchen triggers their association. Ras and GAP Ras is a m e m b e r of a family of small monomeric GTPbinding proteins that act as cellular switches to control a variety of processes - - Ras itself is implicated in the control of cell growth and differentiation, and mutant forms of Ras are associated with human tumors. Ras is active only in the GTP-bound state, which is presumed to interact with as yet unknown downstream effectors. The intrinsic GTPase activity of Ras is low, but it is acceler ated in the presence of GTPase-activating protein (GAP), with which it forms a very tight complex [2]. GAP can only bind to Ras in the GTP-bound state, so that GTP acts as a schatchen in this system, assuming - - as seems likely as its rate of hydrolysis is increased - - that GTP interacts with GAP as well. GTP and GDP both bind to Ras with very high affinity, so why is only GTP a schatchen for Ras and GAP? The answer is simple: the conformation of Ras changes between its GTP-bound and GDP-bound states, and only the Ras~GTP complex has a, surface complementary to GAP. The details of the conformational change in Ras has been determined by conventional and time-resolved crystallography. GTP only binds strongly to Ras in the presence of Mg2 +; the Mg2 + ion is coordinated by one oxygen each from the 13 and 7 phosphates of GTP, and by side chains from loops on the protein surface. When the GTP is hydrolysed to GDP, the 7 phosphate ligand is lost, and the metal ion position and coordination change, triggering a rearrangement in the structure of the two of the loops that furnish ligands to the Mg2 +. These loops are the sites of GAP binding, and only in Ras-GTP are their conformations complementary to GAP. There may be at least one more schatchen in the Ras-GAP system. As the only parts of Ras that change in conformation between the GTP-bound and GDP-bound states are those that bind GAP, and as GAP only binds the active, GTP-bound state of Ras, it is unlikely that the un known downstream effector of Ras can be responding to the Ras structure alone. The only parts of Ras the effector could see are those not covered by GAP, and these do not change in conformation. I suggest that the Ras-GAP complex is the actual switch, and that GTP-bound Ras is a schatchen that promotes the association of GAP with another protein(s) (Fig. 2). Presumably Ras-GTP does this both by changing the conformation of GAP and by also presenting a partial binding surface to the unknown effector, in true double-sided sticky tape fashion. Schatchens galore? There are many other examples of molecular interactions where the schatchen mechanism may be important. Consider, for example, growth factor receptors, where a crucial event in signal transduction appears to be ligandinduced receptor dimerization [11]. But many growth factors are monomeric proteins, so how do they promote dimerization? The three-dimensional structure of

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involve schatchens. Calmodulin is likely to be a schatchen for several systems. Transcription factors clearly function by mediating interactions b e t w e e n proteins, or between proteins and DNA. In all these cases, schatchens pro vide the missing link c o n n e c t i n g proteins that would not otherwise associate in the cellular soup: they are nature's matchmakers, and their mechanisms of action are rapidly being revealed.

References 1. JANINJ, CHOTHt~C: The structure of protein-protein recognition sites. J Biol Chem 1990, 265:16027 16030. 2.

3.

4.

5.

Fig. 2. Action of GTP as a schatchen that allows Ras to bind GAP, and suggested action of GAP as a second schatchen that promotes binding of the Ras-GAP complex to an unknown effector.

the extracellular domain of the h u m a n growth h o r m o n e receptor complexed with h u m a n growth h o r m o n e has b e e n determined [12], and shows h o w a m o n o m e r i c protein schatchen can p r o m o t e the association of two identical receptor subunits. Different sites o n h u m a n growth h o r m o n e are recognized by the same receptor re gion, which is possible because of the flexibility of the receptor site. As in all other cases involving schatchens, the two receptor subunits interact directly as well as via the mediator - - b u t without the d o u b l e sided sticky tape of the schatchen this direct interaction w o u l d be too weak to give a stable dimer. The schatchen mechanism is also likely to b e involved in self/non self discrimination in the i m m u n e system. In cells infected by a virus, peptides derived from viral proteins are presented to cytotoxic T cells b y binding to class I major histocompatibility c o m p l e x (MHC) molecules [13]. The peptides b i n d in a groove formed by two parallel a helices lying o n top of a [3 sheet. The way this complex is recognized by the T cell is not known, b u t an intriguing possibility is that the antigenic peptide modifies the surface of the MHC molecule. The peptide may function as a schatchen, with its e x p o s e d regions also involved in binding to the T cell receptor, giving another example of double-slded sticky tape. C o m p l e m e n t and blood coagulation provide several further examples of p r o t e i n - p r o t e i n recognition that may

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