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Active site-directed protein regulation

Bostjan Kobe & Bruce E. Kemp
St. Vincents Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia

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Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effectors bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.
Protein function in the cell can be modulated in a number of ways, including noncovalent interactions with regulatory factors, covalent modication (for example, phosphorylation, lipidation) or proteolytic cleavage. A widespread mechanism of the direct control of protein function is allosteric regulation, in which effectors bind to regulatory sites distinct from the active site1, usually inducing conformational changes that profoundly inuence the activity. Allosteric effectors generally bear no structural resemblance to their target proteins substrate. This form of regulation explains how the end products of metabolic pathways can act at early steps of the pathway to exert feedback control. Allosteric regulation has been studied intensively for over three decades and is now considered a classical control mechanism of protein function. The term intrasteric regulation was introduced to describe autoregulation of protein kinases and phosphatases by internal sequences resembling substrates (pseudosubstrates) and acting directly at the active site2 (Fig. 1). Several lines of indirect evidence supporting intrasteric regulation were initially obtained using various biochemical approaches. For example, synthetic peptides corresponding to the putative pseudosubstrate regulatory sequences acted as inhibitors; and removal of the regulatory sequences by truncation mutagenesis or limited proteolysis reversed the autoinhibition, as did antibodies directed towards the regulatory sequence3. Unequivocal evidence of intrasteric control, however, has only become available through structural studies of several autoinhibited enzymes; these include the crystal structures of protein kinase A (PKA) with the bound peptide inhibitor4, twitchin kinase5, titin kinase6, calmodulin-dependent protein kinase-1 (CaMK-1)7 and the protein phosphatase calcineurin8. But is the scope of intrasteric control restricted merely to protein kinases and phosphatases? It now emerges that this form of control extends to diverse enzyme classes and beyond, to receptors and protein-targeting domains. Intrasteric control can be used for both autoregulation, either through intramolecular (Fig. 1a) or intermolecular interactions (such as trans interactions between monomers in a oligomer; Fig. 1b), and more general regulation through intermolecular interactions (Fig. 1c). Here we focus on structurally characterized examples in which control is mediated by active sitedirected, intasteric autoregulatory sequences (IARS) (Table 1). Intrasteric interactions typically inhibit protein function. But how is the autoinhibition reversed? Mechanisms used for activation include protein activators or ligands, phosphorylation, proteolysis, reduction of disulphide bonds and combinations of these (Table 1). Intrasteric inhibition requires allosteric control to switch protein function back on (Fig. 1). This combination of intrasteric and allosteric controls provides a powerful and exible mechanism to control some of the most complex cellular processes. (such as myosin-light chain kinase), or directly by calcium (such as plant Ca-dependent protein kinase family; these kinases contain a Ca-binding domain contiguous with the kinase domain). Crystal structures of fragments of twitchin kinase, a giant myosin-associated kinase, show that a carboxy-terminal IARS threads through the active-site cleft between the two lobes of the protein kinase domain, making many contacts with the peptide substrate binding site and other regions essential for catalysis and ATP binding5,9 (Fig. 2a). The binding site of the activator S100A1 has been mapped to one portion of the autoinhibitory sequence, and reverses these interactions to permit substrate access9,10. A similar mechanism of inhibition occurs in the related giant titin kinase; however, here a combination of phosphorylation and calmodulin (CaM) binding (in a site analogous to S100A1-binding site in twitchin) activates the enzyme6. The more distantly related CaMK-1 shows a modied mode of inhibition in which the IARS exits the active site in the opposite direction to that in twitchin kinase7. Although there is no three-dimensional information for other calcium-dependent protein kinases, these are expected to exhibit regulatory mechanisms that are related to those seen in twitchin, titin and CaMK-1. Other protein kinase families predicted to be intrasterically regulated include the protein kinase C family and cGMP-dependent protein kinase, in which the IARS are located amino-terminal to the catalytic domain and the activators are phospholipids and diacyglycerol, and cGMP, respectively3. The crystal structure of the catalytic domain of the insulin receptor tyrosine kinase revealed a more subtle autoinhibitory mechanism with a tyrosine residue bound to the active site11. This
Allosteric site Activator Active site IARS

Catalytic domain Allosteric site Catalytic domains IARS

Active site Activator

Allosteric site

IARS

Active site

Allosteric site

Activator

Active site + Catalytic subunit

IRS

IARS-dependent intrasteric interactions

Protein kinases. Protein phosphorylation is the most common mechanism of cellular regulation, and, to ensure signalling delity, protein kinases (and phosphatases) must be tightly regulated. Many exist in latent forms requiring activation by specic modulators. A large subfamily of kinases are activated by calcium-binding proteins
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Figure 1 Representation of intrasteric regulation. a, Intrasteric regulation of enzymes by intramolecular interactions, through an intrasteric autoregulatory sequence (IARS) that is contiguous with the catalytic domain. The enzyme is maintained in an inactive state through the binding of the IARS in an intramolecular fashion that masks the active site. Allosteric activation by an activatory ligand or protein results in the release of the IARS from the active site. b, Intrasteric regulation of homomeric enzymes by intermolecular interactions, through an IARS in trans. The IARS interacting with one subunit is a part of another subunit. c, Intrasteric regulation of heteromeric enzymes by intermolecular interactions, through an intrasteric regulatory sequence (IRS) on a distinct subunit.
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23 1 Catalytic domain

311 IARS

375 387

19 33 1

120 Catalytic domain

427 452 1

44 5470 IARS Receptor domain

496 529

IARS Regulatory domain

Figure 2 Three-dimensional (3D) structures of representative proteins under intrasteric control through active site-directed autoregulatory sequences. Although IARS can be coarsely identied through limited proteolysis or truncation and site-directed mutagenesis, only 3D structure determination can delineate the contacts with the active site in detail. We dene the IARS as comprising both the sequences interacting with the active site (magenta) and the linker sequence (grey) tethering this segment to other globular domains. a, 3D structure of the twitchin kinase fragment from Aplysia (PDB accession code 1KOB). The IARS is shown in a tube representation, and the rest of the protein in a surface representation (the active-site regions that interact with the IARS are yellow; the active-site regions that do not interact with the IARS are red; the regions not in

the active site but interacting with the IARS are dark blue; and the rest of the surface is light blue). Generated with GRASP46. Bottom, diagram of protein sequence. b, 3D structure of a monomer of phenylalanine hydroxylase (PDB accession code 1PHZ), coloured and drawn as in a. The regulatory domain is shown in green, and the active-site iron atom is shown as a white sphere. All residues lining the active-site cavity have been considered as active-site residues. Only a minor portion of the active site interacts with the IARS. c, 3D structure of importin-a (PDB accession code 1IAL), coloured and drawn as in a. The IARS (magenta) acts as a pseudo-NLS contacting the major NLS-binding site (yellow).

tyrosine may be considered as a transient pseudosubstrate and is ultimately autophosphorylated in response to insulin binding to the extracellular part of the receptor. Phosphorylation of this and two other tyrosine residues results in a rearrangement, allowing access to the active site12. The active site is similarly blocked by a tyrosine in the inactive structure of the serine/threonine kinase mitogenactivated protein (MAP) kinase extracellular signal-regulated kinase (ERK)213. The tyrosine is one of two residues phosphorylated by a distinct upstream kinase to yield the active enzyme14. Protein phosphatases. Autoinhibition of the serine/threonine phosphatase calcineurin (CaN) is caused by an 18-residue sequence C-terminal to the catalytic domain that lies over the substratebinding cleft of the A chain (CaNA)8. It is activated by calcium

binding to the CaM-like B chain (CaNB), and by calcium/CaM binding to CaNA. CaNB and CaM bind to distinct regions in the sequence between the catalytic domain and the autoinhibitory sequence of CaNA. Although three-dimensional information is still lacking, other serine/threonine protein phosphatases are predicted to be intrasterically regulated by both noncatalytic segments and various binding proteins (see above); in some cases, pseudosubstrate sequences have been recognized2,15. The crystal structures of tyrosine phosphatase SHP-2 (ref. 16) and the membrane proximal domain of receptor protein tyrosine phosphatase-a (RPTP-a)17 revealed two further intrasteric mechanisms of inhibition. In SHP-2, the phosphatase active site is blocked by a noncatalytic targeting, Src homology (SH)2 domain

Table 1 Examples of regulation mediated by intrasteric autoregulatory sequences

Protein class
...................................................................................................................................................................................................................................................................................................................................................................

Protein

IARS location

Pseudosubstrate

Activation

Protein kinases

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Twitchin kinase Titin kinase CaMK-1 Insulin receptor kinase MAP kinase ERK2 Calcineurin Receptor PTP-a SHP-2

C-terminal C-terminal C-terminal Internal Internal

Partial Partial Partial Substrate Not apparent

S100A1 binding CaM binding + phosphorylation CaM binding + phosphorylation Phosphorylation Phosphorylation

Protein phosphatases

...................................................................................................................................................................................................................................................................................................................................................................

C-terminal Other subunit in homodimer N-terminal N-terminal N-terminal N-terminal N-terminal N-terminal N-terminal C-terminal N-terminal

Partial Not apparent Not apparent Not apparent Not apparent Not apparent Not apparent Not apparent Not apparent Not apparent Not apparent Yes

CaM binding Phosphorylation Phosphoprotein binding Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis

Proteinases

...................................................................................................................................................................................................................................................................................................................................................................

Serine proteinases Aspartic proteinases Cysteine proteinases Zinc metalloproteinases Multi-molecular assemblies

Metabolic enzymes

...................................................................................................................................................................................................................................................................................................................................................................

Phenylalanine hydroxylase Chloroplast malate dehydrogenase Yeast glycogen phosphorylase

Phenylalanine binding Oxidation Phosphorylation Importin-b binding

Transport receptors Targeting domains

................................................................................................................................................................................................................................................................................................................................................................... ...................................................................................................................................................................................................................................................................................................................................................................

Importin-a

N-terminal

Src, Hck SH2 Src, Hck SH3

C-terminal C-terminal

Yes Yes

Dephosphorylation SH3 ligands

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(the phosphopeptide-binding cleft of the SH2 domain is simultaneously disrupted by this interaction), and activation occurs through phosphopeptide binding by the SH2 domain. In RPTPa, one molecule in the homodimer sterically blocks the active site in the other, forcing it to adopt an open, inactive conformation, and the dissociation is proposed to occur upon phosphorylation. This regulatory mechanism may be physiologically signicant18. There is strong evidence that receptor dimerization is important in signal transduction events19 and intrasteric regulation may be exploited in a number of these examples. Proteinases. Proteinases have diverse biological functions from catabolic digestion to blood coagulation, apoptosis, cleavage of viral precursor proteins to functional units, and pattern formation in multicellular organisms. They are synthesized as inactive precursors (zymogens) to prevent unwanted protein degradation and to enable regulation of proteolytic activity with respect to space and time20. Proteolytic activity is further controlled by specialized proteins that specically inhibit the active enzymes. The structural basis of zymogen autoinhibition was rst shown in crystallographic studies of chymotrypsinogen21 and trypsinogen22,23, and has since been shown in all classes of proteolytic enzyme20. The inhibitory activation segments in zymogens are usually N-terminal extensions of the mature enzymes that sterically block the active site. They can also be insertions within the sequence of the mature enzyme, but, signicantly, they never occur at the C termini, which presumably prevents proteolytic activity during polypeptide synthesis. These segments often have other roles such as folding, stability and/or intracellular sorting. Because the most common mechanism for conversion to active enzymes is limited proteolysis of the inhibitory segment, they are also termed prosegments. The inhibitory segments tend not to mimic the peptide substrates closely, as they have evolved to prevent self-cleavage before the appropriate signal. Consequently, the prosegments of some enzymes (such as papain-like cysteine proteases and pro-stromelysin-1) extend across the active site in the reverse direction to that required for cleavage, whereas the prosegments of others interact with the active site in conformations (such as through a helix in aspartic proteinases) distinct from the extended structures of substrate peptides. Zymogens have various activation mechanisms which may involve accessory molecules, or simply a drop in pH to initiate autocatalysis. The prosegments are usually degraded after activation, presumably to ensure that the conversion is irreversible and that the prosegments do not continue to act as inhibitors. This irreversible activation by proteolysis distinguishes zymogens from other intrasterically regulated proteins, in which the activation is reversible. Mechanisms of inhibition of mature enzymes by protein proteinase inhibitors (such as the bovine pancreatic trypsin inhibitor (BPTI) family, serpins or cystatins) are often distinct from the strategies used by prosegments in zymogens20,24. Some very closely mimic substrates and function by trapping the enzyme into a slow and inefcient cleavage reaction. Metabolic enzymes. Phenylalanine hydroxylase (PAH) is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin, and also by phosphorylation. The crystal structure of the inactive form of PAH shows an N-terminal IARS containing the phosphorylation site which extends from the core of the regulatory domain over the active-site pocket (Fig. 2b)25. Activation may occur by phenylalanine binding to an allosteric site, inducing conformational changes that lead to the removal of the autoinhibitory region from the active site. Phosphorylation in the IARS facilitates this process, but cannot by itself activate the enzyme. Intrasteric control is also responsible for the light regulation of chloroplast nicotinamide adenine dinucleotide phosphate (NADP)-dependent malate dehydrogenase. Light regulation is mediated by thioredoxin-catalysed reduction and re-oxidation of cysteine residues. The structure of the inactive, oxidized protein
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shows a C-terminal IARS that is constrained by a disulphide bond and forced to fold into the active site26,27. In yeast phosphorylase, an N-terminal IARS blocks the active site of the adjacent monomer in the dephosphorylated form; and phosphorylation of the IARS causes it to shift from the active site28. In contrast, mammalian phosphorylase is regulated by a mechanism not involving intrasteric interactions by an IARS29. Transport receptors. Importin-a is the nuclear import factor that recognizes classical monopartite and bipartite nuclear localization signals (NLSs). Mouse importin-a has an N-terminal IARS containing a pseudo-NLS bound to the NLS-binding-site on the large Cterminal receptor domain (Fig. 2c)30. This structure represents the nuclear, low-afnity form of the nuclear import receptor for NLS binding. Conversion to the high-afnity form found in the cytoplasm occurs through binding of the activator protein, importin-b, to a sequence overlapping the pseudo-NLS; importin-b binding markedly alters the conformation of the IARS and presumably exposes the NLS-binding site31. Importin-a is a classical example in which biochemical approaches have led to conicting conclusions regarding intrasteric control. Similar approaches using proteolytic digestion, truncation mutagenesis and synthetic peptides suggested the presence of an autoinhibitory sequence to some authors32,33, but the opposite to others34,35. This underscores, as do studies on protein kinases, the need for three-dimensional information in understanding autoregulation of protein function by intrasteric regulation. Targeting domains. Many signalling proteins contain protein interaction domains, such as SH2 and SH3, that target these proteins to specic substrates. The crystal structures of the inactive forms of c-Src and the related Hck3639 show that the SH2 and SH3 domains are responsible for kinase inactivation through an allosteric mechanism. From another perspective, however, the binding activity of these domains for other binding partners is autoinhibited by kinase sequences through an intrasteric mechanism. The linker connecting the SH2 and the catalytic domain forms a polyproline II helix and binds to the SH3 domain, whereas the SH2 domain binds to a phosphotyrosine in the C-terminal tail of the protein; both interactions are therefore pseudosubstrate-like with respect to these protein interaction domains. Thus, in a highly reciprocal way, the SH2 and SH3 domains regulate the kinase activity while their own targeting activity is simultaneously modulated by the kinase. Tail dephosphorylation and high-afnity ligands for SH2 and SH3 domains would result in kinase activation. A protein kinase domain has been similarly used to autoinhibit the guanylate cyclase activity of the atrial natriuretic peptide receptor, but further structural studies are required to establish the mechanism40.

IARS-independent intrasteric interactions

Alternative intrasteric interactions not mediated by IARSs include active-site modications, such as intrasteric phosphorylation (inhibition by phosphorylation in the active site), and the binding of inhibitory cellular ligands (both proteinaceous and nonproteinaceous) to the active site. In the cyclin-dependent protein kinases, phosphorylation occurs on two residues in the nucleotide-binding loop by the Wee1 kinase41, and in Escherichia coli isocitrate dehydrogenase (IDH) phosphorylation occurs directly on an active-site residue by the bifunctional IDH kinase/phosphatase42. Binding of inhibitors to the active sites and forming intrasteric interactions through intrasteric regulatory sequences is very common in proteinases (BPTI, serpins, cystatins), ribonucleases (mammalian ribonuclease inhibitor, barstar) and protein kinases (protein kinase inhibitor, PKA regulatory subunits).

Conclusions
Our examples of intrasteric regulation conrm predictions that pseudosubstrates act at the active site, and expectations that the contacts made by the inhibitory segments are more complex and do not merely mimic the substrates (compare the yellow and red
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regions in Fig. 2). In cases such as zymogens, it is advantageous for the inhibitory segment to be more pseudo than substrate, to prevent unwarranted activation. Although unequivocal evidence of intrasteric control can only be provided though structural studies, other evidence, obtained, for example, by truncation mutagenesis, limited proteolysis, synthetic peptides corresponding to the regulatory sequences and antibodies directed towards the regulatory sequences, can shed further light on the regulatory process. Structural information alone is not sufcient evidence for a biologically relevant regulatory mechanism; intrasteric interactions through C-terminal sequences observed in fragments of chaperones DnaK and Hsc70 may be caused by the truncated protein constructs used43,44, and similar interactions of a C-terminal peptide with an adjacent molecule in the crystals of protein farnesyltransferase45 may be driven by the crystallization process rather than by native regulation. In many cases, the interactions of the inhibitory segment with the rest of the protein do not appear particularly strong. Many proteinase prosegments or portions thereof have high-temperature factors suggesting high mobility, and in importin-a regions anking the autoinhibitory sequence are disordered. It seems reasonable that this is important in facilitating the activation process, and the nature of the interactions formed by the IARS must clearly reect the mechanism of activation. As an example, a prominent feature of the pH-activated zymogen prosegment interactions is the critical role of salt bridges. Because the thermodynamics and kinetics of inhibition and activation processes in intrasterically regulated proteins are generally poorly understood, studies of these parameters accompanied by further structural studies of both the inhibited and activated states should represent some of the most important future research directions. The examples show that intrasteric regulation through IARS can occur in monomeric systems (in which the IARS and the catalytic regions are located within the same polypeptide chain; Fig. 1a) and homomeric systems (in which the IARS is located either in the same monomer as the catalytic domain (cis, intramolecular interaction) or within a different monomer of the oligomer (trans, intermolecular interaction; Fig. 1b)) (Table 1). Intrasteric interactions can also be achieved in other ways, such as by regulatory sequences contained within transient protein subunits (such regulation can be thought of as analogous to the regulation of homomeric proteins in trans, but instead occurring in heteromeric systems; Fig. 1c); covalent modications of the active-site residues (for example, intrasteric phosphorylation); and binding of cellular effectors to the active site. Intrasteric regulation may be a widespread control mechanism that is prominent in the regulation of cellular functions; it will undoubtedly be found in many more proteins as the repertoire of protein three-dimensional structures expands. Intrasteric control is typically accompanied by allosteric control to turn protein function on; it is this combination of intrasteric and allosteric controls that provides the exibility to achieve almost any level of regulation of protein function that may be required.
1. Monod, J., Changeux, J. P. & Jacob, F. Allosteric proteins and cellular control systems. J. Mol. Biol. 6, 306329 (1963). 2. Kemp, B. E. & Pearson, R. B. Intrasteric regulation of protein kinases and phosphatases. Biochim. Biophys. Acta 1094, 6776 (1991). 3. Kemp, B. E. et al. in Protein Kinases (ed. Woodgett, J. R.) 3067 (IRL, Oxford, 1994). 4. Knighton, D. R. et al. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 414420 (1991). 5. Hu, S.-H. et al. Insights into autoregulation from the crystal structure of twitchin kinase. Nature 369, 581584 (1994). 6. Mayans, O. et al. Structural basis for activation of the titin kinase domain during myobrillogenesis. Nature 395, 863869 (1998). 7. Goldberg, J., Nairn, A. C. & Kuriyan, J. Structural basis for the auto-inhibition of calcium/calmodulindependent protein kinase I. Cell 84, 875887 (1996). 8. Kissinger, C. R. et al. Crystal structures of human calcineurin and the human FKBP12-FK506calcineurin complex. Nature 378, 641644 (1995). 9. Kobe, B. et al. Giant protein kinases: domain interactions and structural basis of autoregulation. EMBO J. 15, 68106821 (1996). 10. Heierhorst, J. et al. Ca2+/S100 regulation of giant protein kinases. Nature 380, 636639 (1996). 11. Hubbard, S. R., Wei, L., Ellis, L. & Hendrickson, W. A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372, 746754 (1994). 12. Hubbard, S. R. Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog. EMBO J. 16, 55725581 (1997). 13. Zhang, F., Strand, A., Robbins, D., Cobb, M. H. & Goldsmith, E. J. Atomic structure of the MAP kinase ERK2 at 2.3 A resolution. Nature 367, 704711 (1994). 14. Canagarajah, B. J., Khokhlatchev, A., Cobb, M. H. & Goldsmith, E. J. Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859869 (1997). 15. Barford, D., Das, A. K. & Egglof, M. P. The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu. Rev. Biophys. Biomol. Struct. 27, 133164 (1998). 16. Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M. J. & Shoelson, S. E. Crystal structure of the tyrosine phosphatase SHP-2. Cell 92, 441450 (1998). 17. Bilwes, A. M., den Hertog, J., Hunter, T. & Noel, J. P. Structural basis for inhibition of receptor proteintyrosine phosphatase-a by dimerization. Nature 382, 555559 (1996). 18. Jiang, G. et al. Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase- . Nature 401, 606610 (1999). 19. Weiss, A. & Schlessinger, J. Switching signals on or off by receptor dimerization. Cell 94, 277280 (1998). 20. Khan, A. R. & James, M. N. G. Molecular mechanisms for the conversion of zymogens to activate proteolytic enzymes. Protein Sci. 7, 815836 (1998). 21. Freer, S. T., Kraut, J., Robertus, J. D., Wright, H. T. & Xuong, N. H. Chymotrypsinogen: 2.5-angstrom crystal structure, comparison with alpha-chymotrypsin, and implications for zymogen activation. Biochemistry 9, 19972009 (1970). 22. Kossiakoff, A. A., Chambers, J. L., Kay, L. M. & Stroud, R. M. Structure of bovine trypsinogen at 1.9 A resolution. Biochemistry 16, 654664 (1977). 23. Fehlhammer, H., Bode, W. & Huber, R. Crystal structure of bovine trypsinogen at 18 A resolution. II. Crystallographic renement, rened crystal structure and comparison with bovine trypsin. J. Mol. Biol. 111, 415438 (1977). 24. Bode, W. & Huber, R. Natural protein proteinase inhibitors and their interactions with proteinases. Eur. J. Biochem. 204, 433452 (1992). 25. Kobe, B. et al. Structural basis of autoregulation of phenylalanine hydroxylase. Nature Struct. Biol. 6, 442448 (1999). 26. Carr, P. D., Verger, D., Ashton, A. R. & Ollis, D. L. Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction. Structure 7, 461475 (1999). 27. Johansson, K. et al. Structural basis for light activation of a chloroplast enzyme: the structure of sorghum NADP-malate dehydrogenase in its oxidized form. Biochemistry 38, 43194326 (1999). 28. Lin, K., Hwang, P. & Fletterick, R. J. Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylase. Structure 5, 15111523 (1997). 29. Johnson, L. N., Barford, D., Owen, D. J., Noble, M. E. & Garman, E. F. From phosphorylase to phosphorylase kinase. Adv. Second Messenger Phosphoprotein Res. 31, 1128 (1997). 30. Kobe, B. Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha. Nature Structur. Biol. 6, 388397 (1999). 31. Cignolani, G., Petosa, C., Weis, K. & Muller, C. W. Structure of importin-beta bound to the IBB domain of importin-alpha. Nature 399, 221229 (1999). 32. Moroianu, J., Blobel, G. & Radu, A. The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence. Proc. Nat. Acad. Sci. USA 93, 65726576 (1996). 33. Conti, E., Uy, M., Leighton, L., Blobel, G. & Kuriyan, J. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell 94, 193204 (1998). 34. Gorlich, D., Henklein, P., Laskey, R. A. & Hartmann, E. A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. EMBO J. 15, 18101817 (1996). 35. Weis, K., Ryder, U. & Lamond, A. I. The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import. EMBO J. 15, 18181825 (1996). 36. Xu, W., Harrison, S. C. & Eck, M. J. Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595602 (1997). 37. Sicheri, F., Moare, I. & Kuriyan, J. Crystal structure of the Src family tyrosine kinase Hck. Nature 385, 602653 (1997). 38. Williams, J. C. et al. The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions. J. Mol. Biol. 274, 757775 (1997). 39. Moare, I. et al. Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement. Nature 385, 650653 (1997). 40. Chinkers, M. & Garbers, D. L. The protein kinase domain of the ANP receptor is required for signalling. Science 245, 13921394 (1989). 41. Morgan, D. O. Principles of CDK regulation. Nature 374, 131134 (1995). 42. Hurley, J. H., Dean, A. M., Sohl, J. L., Koshland, D. E. Jr & Stroud, R. M. Regulation of an enzyme by phosphorylation at the active site. Science 249, 10121016 (1991). 43. Wang, H. et al. NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction. Biochemistry 37, 79297940 (1998). 44. Morshauer, R. C. et al. High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70. J. Mol. Biol. 289, 13871403 (1999). 45. Park, H.-W., Boduluri, S. R., Moomaw, J. F., Casey, P. J. & Beese, L. S. Crystal structure of protein farnesyltransferase at 2.25 Angstrom resolution. Science 275, 18001804 (1997). 46. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281296 (1991).

Acknowledgements
We thank J. Heierhorst, I. Jennings and other members of the protein research groups at St. Vincents Institute for discussions; T. Teh for comments on the manuscript; and P. Carr for unpublished data. The work was supported by the National Health and Medical Research Council, Wellcome Trust, Australian Research Council and National Heart Foundation; B.K. is a Wellcome Senior Research Fellow in Medical Science in Australia; B.E.K. is an NHMRC Fellow. Correspondence and requests for materials should be addressed to B.K. (e-mail: b.kobe@medicine.unimelb.edu.au).
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