Laboratory of Molecular Biophysics
Laboratory Journal 2003
Louise N. Johnson

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Louise N. Johnson

Structural studies on protein kinases and regulatory molecules of the cell cycle

Introduction

We are interested in the structural basis of the recognition processes that control biological interactions, especially those that mediate control by phosphorylation. Our work in the year 2002/2003 has focussed on the regulatory proteins of the cell cycle, that include CDK2/cyclin A, CDK2/cyclin E, CDK7/cyclin H, polo-like kinase and those proteins that mediate interactions of ubiquitinated proteins with the proteasome. We have begun a new programme with CDK9/cyclin T in collaboration with Pharmacia (Nerviano), the cyclin dependent protein kinase that plays a key role in transcription. We continue our special interest with phosphorylase kinase and its regulation by calcium/calmodulin. Our major technique is protein crystallography, which for some problems is combined with electron microscopy (see research report by Catherine Venien Bryan).

1 Cell cycle proteins

1.1 Polo-like kinase

Robert Cheng, Ed Lowe and John Sinclair in collaboration with Erich A. Nigg (Max Planck Institute for Biochemistry, Martinsried)

Polo-like kinases (Plks) are important regulators of cell cycle progression during mitosis. Named after the polo gene of Drosophila melanogaster, Plks are involved in the assembly and dynamics of the mitotic spindle apparatus, in the activation and inactivation of CDK/cyclin complexes and in the phosphorylation of numerous substrates that control progression through M phase.

Plk1 is a 603 amino acid protein that comprises an N-terminal kinase domain and a C-terminal regulatory polo-box domain (PBD). The PBD contains two sequences of about 80 amino acids that are conserved in all polo-like kinases. Cellular studies have shown that the PBD is important for sub-cellular localisation and is also an autoinhibitory domain. In an important result in 2003, Elia et al. (1) showed that the PBD recognised a phospho-peptide motif from the regulatory domain of the phosphatase CDC25C that contained the sequence Ser.phosphoThr.Pro.

This year we have cloned, expressed, purified and crystallised the PBD (residues 367-603) of human Plk1. The structure was solved by SAD phasing using a seleno-methionine derivative collected at ESRF station ID29 and with high-resolution native data collected to 2.2 Å resolution at Elettra. The crystal structure of the PBD complexed with the cognate peptide M.Q.S.pT.P.L allowed the peptide-binding region to be identified (Figure 1) (2). The phospho-peptide binds at a site between the two polo boxes. It makes a short anti-parallel

sheet connection to the first

-strand

1 and critical contacts to residues Trp414, Leu490, His538 and Lys540. Most of these residues had been shown to be important for biological activity through mutational studies. The results provide an explanation for phospho-peptide recognition and create the basis for new functional studies.

The structural studies are continuing with the aim of understanding the kinase domain and the way in which the PBD regulates kinase activity.

Figure 1. The structure of the Polo-like kinase polo-box domain in complex with a phospho-peptide. Left: a schematic diagram showing the Plk1 PBD and the bound phospho-peptide MQSpTPL (magenta). The N-terminal extension and the PBD1 are shown in dark blue and the PBD2 in cyan. Right: The van der Waals surface of the PBD with the hydrophobic potential calculated by GRID displayed. The atoms of the phospho-peptide are superimposed. The view is 90° to that shown on the left. Lower: a stereo diagram of the interactions between the phospho-peptide and the residues at the binding site. The view is similar to that shown for the van der Waals surface. Drawings were made with AESOP (Martin Noble, unpublished work). (from Cheng et al. (3).

1.2 CDK2/cyclin A recruitment site

Robert Cheng, Vicky Skamnaki, Ed D. Lowe, Nick R. Brown, Luke Kontogiannis in collaboration with Jim McDonnell

Progression through the cycle is driven by the sequential activities of different cyclin dependent protein kinases (CDKs) that require association with their respective cyclins and phosphorylation for activation. Selection of the right substrates is a key event in the organisation of both the temporal and spatial events that characterise the complex processes of cell growth and cell division.

During each stage of the cycle, the CDKs recognise specific substrates. The obligatory local epitope that is recognised by CDK2/cyclin A is Ser/Thr.Pro.X.Lys/Arg where Ser or Thr are the residues phosphorylated (3). A decade ago the substrates p107 and p130 were found to have an interaction domain that promoted their stoichiometric association with CDK2/cyclin E or CDK2/cyclin A and which is shared with the CDK2 inhibitors p21 and p27. It is now recognised that many other CDK2 substrates contain a site that is remote from the site of phosphorylation and which contains the signature motif RXL or KXL. The R(K)XL motif is recognised by a specific hydrophobic binding site on cyclin A that is located over 40Å from the catalytic site. Studies with other protein kinases, such as the MAP kinase family, PDK1 and glycogen synthase kinase have shown that remote recruitment or docking sites are also an essential feature of several kinases substrate recognition.

How does the recruitment site govern events at the catalytic site? There are three (or at least three) possibilities illustrated in Figure 2. Firstly the recruitment site may serve to localise the kinase on the substrate and hence to increase the local substrate concentration (Figure 2a). In view of the importance of substrate targeting in protein kinases and the potential for exploiting this phenomena in anti-cancer drug design (4), we wished to understand the phenomena in structural terms.


Figure 2. A schematic diagram showing how recruitment (docking) sites might contribute to substrate selection and catalysis. (a) The recruitment site localises the substrate in the vicinity of the enzyme through formation of transient complexes. The effects are solely entropic and there is no direct connection between the sites. (b) The recruitment site binds to the substrate and tethers it so that the substrate has a smaller conformational space to explore in order to reach the catalytic site. The recruitment may take place through a site on the enzyme or through a site on a regulatory subunit or domain. (c) The recruitment site not only localises the substrate and tethers it but there is a direct stereochemical route between the docking site and the catalytic site that involves other parts of the substrate, regulatory subunit and enzyme.

Last year we reported on structural studies that elucidated the specificity for additional sequences around the RXL (or KXL) site by examining binding of peptides derived from pRb, p107, p27, E2F and p53 (5).

This year we have examined the enzyme kinetics of the action of phospho-CDK2/cyclin A on a 30 amino acid peptide derived from the CDC6 protein, the protein involved in the formation of the pre-replication complex that is required for initiation of DNA replication. CDC6, a natural substrate, contains the Arg of the RXL motif only 20 amino acids C-terminal from the Ser of the SPXK sequence of the substrate phosphorylation site (Table 1). Comparison of the kinetic parameters with a peptide substrate containing no recruitment site with the CDC6 peptide shows that the consequence of the recruitment site is to produce a six-fold enhancement of the specificity constant kcat/Km. Studies in which the two peptides were not linked covalently show no effect on the kinetic parameters. The results support a direct role of the recruitment site on catalysis.

The structure of phospho-CDK2/cyclin A co-crystallised with the CDC6 peptide showed binding at the catalytic site for the sequence HHASPRK (residues 71-77), a gap in electron density, and then binding for the sequence 89-100 at the recruitment site (Figure 3). There were extensive interactions for the whole of the twelve amino acid length peptide bound at the recruitment site. In order to investigate the temporal aspects of binding at the two sites and possible transient interactions in the intervening region between the recruitment and catalytic site, HSQC NMR experiments are in progress.

Figure 3. Binding of the CDC6 peptide to phospho-CDK2/cyclin A. CDK2 is in light blue, cyclin A is in pink, AMPPNP is in yellow, pThr160 in green, peptide in white and electron density in blue. The peptide binds at the substrate site in pCDK2 and at the recruitment site in cyclin A. (From Cheng MSc thesis, University of Oxford, 2003).

1.3 CDK2/cyclin E

Elena Dubinina, Vicky Skamnaki and Atlanta Cook

Cyclin E becomes active at G1/S boundary. CDK2/cyclin E appears to be a rather more selective enzyme than CDK2/cyclin A phosphorylating fewer substrates or phosphorylating them less well than CDK2/cyclin A. The recruitment substrate-binding site on cyclin E is identical in amino acid sequence to that of cyclin A and appears to play a similar role. Since CDK2 is the same in the CDK2/cyclin A and CDK2/cyclin E complexes why does the cell need both cyclin A and cyclin E? In an astonishing result this year Geng et al (6) working with knockout mouse cells showed that cyclin E is not required for cell proliferation under conditions of continuous cell cycling but is required to allow cells to re-enter the cell cycle from the quiescent G0 phase. There appears to be a key step involved in the incorporation of MCM proteins into DNA replication origins, which requires cyclin E. Importantly the work of Coverley et al (7) had already shown that cyclin E must act before cyclin A to allow normal DNA replication. We wish to understand the different cellular regulatory roles of cyclin E and cyclin A through structural analysis. Cyclin E is over-expressed in human breast cancers and cyclin E deficient cells are relatively resistant to oncogenic transformation. Hence there is an interest in developing selective inhibitors.

Cyclin E has been expressed in Sf9 insect cells using a baculoviral construct kindly given by David Morgan (UCSF) and the complex with phospho-CDK2 purified. Kinetic studies with pCDK2/cyclin E and with pCDK2 in complex with a truncated version of cyclin E prepared by limited digestion with elastase show that CDK2/cyclin E is as good an enzyme as CDK2/cyclin A with model peptide systems and that the effects of the recruitment site are similar for CDK2/cyclin A and CDK2/cyclin E (Table 1). One substrate that is a good substrate for CDK2/cyclin A but not CDK2/cyclin E is nuclear lamin B (8). We have developed an E. coli expression system for nuclear lamin and various fragments of this protein and have confirmed this result with purified enzymes. Crystallisation studies of pCDK2/cyclin E have yielded small needle like crystals but these need to be slightly larger before we can contemplate data collection.

Table 1. Kinetic parameters for CDK2/cyclin A and CDK2/cyclin E against peptide substrates.
Enzyme	Substrate	K^cat(peptide) s^-1	K^m(peptide) µM	K^cat/K^m s^-1µM^-1
CDK2/A^a	HHASPRK	34.2 ± 2.5	793 ± 89	0.046
CDK2/A^a	CDC6 modified peptide^c HHASPRKQGKKENGPPHSHTLKGRRLVFDN	18.9 ± 2.4	74 ± 24	0.25
CDK2/A^b	HHASPRK	68.2 ± 11.7	802 ± 230	0.085
CDK2/A^b	(HHASPRK) + (HTLKGRRLVFDN)	64.5 ± 0.8	865 ± 20	0.074
CDK2/E	HHASPRK	30.5 ± 5.6	766 ± 102	0.039
CDK2/_Etruncated	HAASPRK	53.2 ± 4.5	1005 ± 127	0.053
CDK2/_Etruncated	CDC6 modified peptide^c HHASPRKQGKKENGPPHSHTLKGRRLVFDN	22.6 ± 1.7	181 ± 31	0.125
CDK2/E	HHASPRK	19.8 ± 0.7	503 ± 39	0.039
CDK2/E	(HHASPRK) + (HTLKGRRLVFDN)	18.7 ± 1.5	554 ± 98	0.033

^{a, b} Denote different preparations of pCDK2/cyclin A.
^c The CDC6 peptide has been modified from the natural peptide to contain the optimal sequence HHASPRK around the site of phosphorylation. The natural sequence in CDC6 is PPCSPPK.

1.4 CDK Activating Kinase (CAK)

Graziano Lolli, Ed D. Lowe and Nick R. Brown

CAK is a positive regulator of CDK1, CDK2 and CDK4. The kinase phosphorylates CDKs on the threonine residue in the activation segment (Thr160 in CDK2). CAK is itself a member of the CDK family and is composed of CDK7, cyclin H and an assembly protein Mat1. CDK7 raises intriguing questions concerning substrate recognition. CDK7/cyclin H/Mat1 ternary complex also exists as an integral component of the transcription/DNA repair nine-protein complex TFIIH. In TFIIH CDK7 complex phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II, which allows initiation of transcription and generation of short RNA transcripts. TFIIH specifically associates with U1 snRNA, a core splicing component, through cyclin H providing a link between transcription and RNA processing (9).

With generous gifts of new viruses from Robert Fisher (Memorial Sloan Kettering Cancer Centre, New York), Ernst Laue (Department of Biochemistry, Cambridge) and Erich Nigg (Max Planck Institute, Martinsried) we are developing expression strategies to produce proteins for crystallisation. Human CDK7 has been expressed and purified and shown by mass spectrometry to be 30% bi-phosphorylated and 70% mono-phosphorylated. The protein has been crystallised and data from these crystals (dimensions ~ 80 x 30 x 30 mm)) were collected at the microfocus beamline ID13 at ESRF to 3 Å resolution. The structure has been solved by molecular replacement to give the first insights into this new CDK regulatory system.

1.5 Regulators of transcription (CDK9/cyclin T)

Graziano Lolli and Ed Lowe

The cyclin dependent protein kinase, CDK9/cyclin T, regulates the elongation phase of RNA transcription during the cell cycle. In concert with CDK7/cyclin H/Mat1, CDK9/cyclin T, further phosphorylates the CTD at sites in a heptad 52 repeat motif of RNA polymerase II and moves transcription to a production stage. CDK9/cyclin T is also involved in the trans-activation of HIV transcription through the recruitment of the quaternary complex of CDK9/cyclin T, TAT and TAR, a short RNA generated by initiation of transcription from the HIV promoter, to RNA polymerase II (10).

Figure 4 shows a schematic view of the dual roles of CDK7 and CDK9.

Schematic view of the dual roles of CDK7 and CDK9.

Working from constructs developed at Pharmacia, Nerviano, soluble expression of active CDK9/cyclin T has been produced through dual virus infection in insect cells. The complex has been crystallised to yield very tiny crystals that diffract to 7 Å resolution. Work is in progress to further characterise the complex and to improve the crystals in order to provide a structural basis for the biological properties of this important CDK.

1.6 Studies with Ubiquitin Domain Proteins (UDPs)

Nick R. Brown, Ed Lowe in collaboration with Jane Endicott, Martin Noble, Jean-Francois Trempe, Hideki Kobayashi (Kyushu University, Japan) and Colin Gordon (Edinburgh, UK)

Ubiquitin (Ub) is used as a signal molecule for controlled targeting of proteins to the proteasome and in other signalling processes. A cascade of enzymes catalyses the selective attachment of Ub proteins: an ATP-dependent Ub-activating enzyme (E1) in combination with an Ub-conjugating enzyme (E2) permits the covalent attachment of Ub to another Ub molecule or to a substrate protein; the latter being specified by a third family of enzymes, the Ub ligases (E3).

Dsk2 is a member of a family of proteins that typically contain both an N-terminal Ub-like (UBL) domain and a C-terminal Ub-associated (UBA) domain. UBA domains are one of a number of Ub binding families, which also include the Ub interacting motif (UIM) and the Coupling of Ub conjugation to ER degradation (CUE) domains. Recent structural data (primarily NMR) have been obtained for all 3 types of module interacting with mono-Ub. We wish to extend these data with structural results for Dsk2 UBA interacting not only with mono-Ub but also for di-Ub and tetra-Ub, enabling a comparative molecular description for these protein/ protein interactions.

The bifunctional binding properties of proteins such as Dsk2 have suggested a model for their function whereby the UBA domain binds the poly-Ub moiety of an ubiquitinated substrate and the UBL domain binds to specific subunits of the proteasome, thus delivering the substrate protein to the proteasome for subsequent degradation. Recent work by others have localised the regions within the Rpn1 proteasomal subunit responsible for UBL binding and have guided our attempts to produce active fragments bound to UBL domains for crystal structure solution. Expression of Rpn1 fragments CD (residues 417-737) and CD

(residues 417-628) as GST-fusions has been achieved.

As reported last year, we have a 1.3Å structure of the Dsk2 UBL domain and have grown crystals and collected 2.5Å native data for the Dsk2 UBA domain. The latter structure has proved elusive and although several different crystal morphologies have been grown, structure solution has been complicated by the problem of twinning in seleno-methionine crystals. We hope to solve these problems in the not too distant future. We have found that Dsk2 UBA is able to form a complex with mono-Ub, which is stable during gel filtration. Crystallisation trials using the TECAN robot of a Dsk2 UBA/ mono-Ub complex have yielded several conditions producing needle crystals and one further condition producing very small three dimensional crystals of 5-10µm. Encouragingly the latter crystals diffracted to more than 5Å at the ESRF microfocus beamline ID13 despite their extremely small size.

The availability of recombinant E1 in-house, has meant that we are now able to produce poly-Ub chains. A recombinant baculovirus encoding histidine-tagged mouse Ub-activating enzyme (a kind gift of Kazuhiro Iwai, Osaka Japan) has now proven to be a good source of E1 activity. Fractions from a Ni-NTA column promote the assembly of Ub into chains when mixed with recombinant human CDC34 (supplied by Jan Gruber, LMB) and mono Ub. Preliminary experiments have focussed on optimising the yields of di-Ub at the expense of longer poly-Ub chains and milligram quantities have now been purified using Superdex SD30 chromatography.

2 Regulation of phosphorylase kinase

Atlanta Cook, Vicky Skamnaki and Dietbert Neumann

Phosphorylase kinase plays a central role in coordinating cellular signals in the regulation of glycogen metabolism, from hormone responses via cAPK and through calcium signalling. The kinase converts inactive glycogen phosphorylase b to the active glycogen phosphorylase a through specific phosphorylation of residue Ser14. The kinase is a large hexadecameric holoenzyme made up of four different subunits:

and

. The

chain contains the kinase domain and a C-terminal regulatory region. The

subunit is an intrinsic molecule of calmodulin (CaM) that binds to the regulatory region of the

chain and confers calcium sensitivity on the enzyme. The

and

subunits also regulate the

subunit by incorporating signals from phosphorylation, the binding of extrinsic Ca²⁺/calmodulin and metabolite binding.

Following structure determinations of the kinase domain (11) and an electron microscope image at 22 Å resolution of the holoenzyme in complex with glycogen phosphorylase (12), we now wish to understand the molecular details of the regulation of kinase activity. We are currently attempting to co-express the holoenzyme

complex in E. coli using the strategy adopted by Dietbert Neumann and colleagues in Zurich for co-expression of the three subunits of AMP dependent protein kinase (13). Following gifts of clones of the

and

subunits of both muscle and liver phosphorylase kinase from Manfred Kilimann (Bochum and Uppsala), expression of small amounts of the ternary

complex has been achieved. The complex is active and activity is calcium dependent as expected. These studies are continuing to produce the quaternary complex for electron microscope studies.

Following the development of a new expression system for the kinase domain of the catalytic

subunit (Lab Journal 2001/2002), we have investigated further the selective inhibitory properties of the staurosporine analogue KT5720. Kinetic analysis shows indeed that it is a potent inhibitor of phosphorylase kinase (Ki = 20 nM) and much less potent for pCDK2/cyclin A (no inhibition at 300 nM). Crystallographic binding studies are in progress.

The

subunit of phosphorylase kinase is an intrinsic molecule of CaM that forms a complex with the

subunit. As in many Ca²⁺/CaM-dependent protein kinases the

subunit C-terminal autoinhibitory region overlaps with a CaM binding domain, and consists of a sequence that can form a basic, amphipathic helix. Two peptides of 25 amino acids in length from this region were identified as CaM binding peptides through their ability to inhibit the Ca²⁺/calmodulin-dependent activation of myosin light chain kinase (14). The peptides, PhK5 (residues 342-366) and PhK13 (residues 302-326) appear to bind simultaneously to CaM in different binding modes (15, 16). The expression system for calmodulin that utilises the PROTet vector produces significant amounts of calmodulin. Analytical gel filtration experiments indicated that CaM/PhK5 complexes were stable but CaM/PhK13 complexes were not.

We have turned to NMR in order to characterise the interactions of the peptides with calmodulin. HSQC experiments were carried out with ¹⁵N labelled calmodulin and titrating in PhK5 and PhK13 in the presence of Ca²⁺. The PhK5 titrations, using increments of 10-20 %, showed a large number of chemical shift changes on titration. In addition, the binding of the peptide showed slow exchange kinetics consistent with the nanomolar affinity constants reported in the literature. As CaM has previously been assigned, most of the chemical shifts could be identified. In contrast, titration of PhK13 into CaM did not cause any chemical shift changes, demonstrating that PhK13 does not interact with CaM.

To further characterise the Ca²⁺/CaM/PhK5 complex, data points for T1 and T2 were collected with a 600 MHz spectrometer. Hydrodynamic parameters were fitted to the longitudinal and transverse relaxation rates using ROTDIF (17). This approach took advantage of the relatively large database of CaM/peptide structures in the PDB and used a series of seven known complexes as input models for the relaxation data. The hydrodynamic analysis showed that the relaxation data for Ca²⁺/CaM/PhK5 has the best fit with, and is therefore most closely related to three out of the seven complexes that were analysed, namely the Ca²⁺/CaM/eNOS, Ca²⁺/CaM/CaMKI and Ca²⁺/CaM/smMLCK complexes (Ms in preparation). These three structures share a 1-14 motif for the placement of anchoring residues along their peptides.
References

Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates, Science 299, 1228-31.
Cheng, K.-Y., Lowe, E. D., Sinclair, J., Nigg, E. A., and Johnson, L. N. (2003) The crystal structure of the human polo-like kinase polo-box domain and its complex with a phosphopeptide, EMBO J. In press.
Brown, N. R., Noble, M. E. M., Endicott, J. A., and Johnson, L. N. (1999) The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases, Nature Cell Biology 1, 438-443.
Chen, Y.-N. P., Sharma, S. K., Ramsey, T. M., Liang, L., Martin, M. S., Baker, K., Adams, P. D., Bair., K. W., and Kaelin, W. G. (1999) Selective killing of transformed cells by cyclin/cyclin dependent kinase 2 antagonists, Proc. Natl. Acad. Sci. USA 96, 4325-4329.
Lowe, E. D., Tews, I., Cheng, K. Y., Brown, N. R., Gul, S., Noble, M. E., Gamblin, S. J., and Johnson, L. N. (2002) Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A, Biochemistry 41, 15625-34.
Geng, Y., Yu, Q., Sicinska, E., Das, M., Schneider, J. E., Bhattacharya, S., Rideout, W. M., Bronson, R. T., Gardner, H., and Sicinski, P. (2003) Cyclin E ablation in the mouse, Cell 114, 431-43.
Coverley, D., Laman, H., and Laskey, R. A. (2002) Distinct roles for cyclins E and A during DNA replication complex assembly and activation, Nat Cell Biol 4, 523-8.
Horton, L. E., and Templeton, D. J. (1997) The cyclin box and C-terminus of cyclins A and E specify CDK activation and substrate specificity, Oncogene 14, 491-8.
Kwek, K. Y., Murphy, S., Furger, A., Thomas, B., O'Gorman, W., Kimura, H., Proudfoot, N. J., and Akoulitchev, A. (2002) U1 snRNA associates with TFIIH and regulates transcriptional initiation, Nat Struct Biol 9, 800-5.
Romano, G., Kasten, M., De Falco, G., Micheli, P., Khalili, K., and Giordano, A. (1999) Regulatory functions of Cdk9 and of cyclin T1 in HIV tat transactivation pathway gene expression, J Cell Biochem 75, 357-68.
Lowe, E. D., Noble, M. E. M., Skamnaki, V. T., Oikonomakos, N. G., Owen, D. J., and Johnson, L. N. (1997) The crystal structure of a phosphorylase kinase peptide substrate complex: kinase substrate recognition, EMBO J. 16, 6646-6658.
Venien-Bryan, C., Lowe, E. D., Boisset, N., Traxler, K. W., Johnson, L. N., and Carlson, G. M. (2002) Three-dimensional structure of phosphorylase kinase at 22 Å resolution and its complex with glycogen phosphorylase, Structure 10, 33-41.
Neumann, D., Woods, A., Carling, D., Wallimann, T., and Schlattner, U. (2003) Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli, Protein Expr Purif 30, 230-7.
Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1989) The g subunit of skeletal muscle phosphorylase kinase contains two non-contiguous domains that act in concert to bind calmodulin., J. Biol. Chem. 264, 17156-17163.
Trewella, J., Blumenthal, D. K., Rokop, S. E., and Seeger, P. A. (1990) Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit phosphorylase kinase, Biochemistry 29, 9316-9324.
Juminaga, D., Albaugh, S. A., and Steiner, R. F. (1994) The interaction of calmodulin with regulatory peptides of phosphorylase kinase, J. Biol. Chem. 269, 1660-1667.
Fushman, D., Xu, R., and Cowburn, D. (1999) Direct determination of changes of interdomain orientation on ligation: use of the orientational dependence of 15N NMR relaxation in Abl SH(32), Biochemistry 38, 10225-30.