Human LIMK1-ATPgammaS-CFL1 Complex

Target ID:

XX02LIMK1A

Deposition Date:

Tuesday 31st May 2016

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-protein

Download Coordinates:

5L6W

Authors:

Salah, E., Mathea, S., Oerum, S., Newman, J.A., Tallant, C., Adamson, R., Canning, P., Beltrami, A., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Knapp, S., Bullock, A.N.

Site:

Oxford

5L6W

tabs

Structure Details

Human LIM kinase 1 (LIMK1) in complex with CFL1 and ATPγS

The LIM kinase family comprises LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) which show 50% sequence identity (1). LIMK1 and LIMK2 share an unique domain organization containing two N-terminal LIM domains, a PDZ domain, a proline/serine-rich domain and a C-terminal kinase domain. Both proteins are expressed widely in embryonic and adult tissues. The two kinases have overlapping functions, but appear non-redundant. Knockout studies in mice show that LIMK1 is required for development of the central nervous system (2), whereas LIMK2 knockout impairs the activity of testicular germ cells (3). In humans, LIMK1 loss is part of a chromosomal microdeletion associated with Williams syndrome, a developmental disorder characterized by neurocognitive and social-behavioral abnormalities (4, 5). The LIM kinases also contribute to tumour cell invasion and metastasis, which is reversed by gene silencing or dominant negatives, suggesting that LIM kinases are important targets for drug discovery (6). In particular, LIMK1 overexpression is found in malignant melanoma, breast and prostate cancers, as well as most tumour cell lines.

The effects of LIM kinase activity on cell morphology and motility are thought to be determined by their regulation of the actin cytoskeleton (7). The LIMKs signal downstream from Rho GTPases and are activated by phosphorylation of the activation loop by upstream kinases, including Rho kinase (ROCK), PAK1/2/4 and MRCKα. The best characterized LIMK substrates are cofilin1 (non-muscle cofilin, CFL1), cofilin2 (muscle cofilin, CFL2) and destrin (actin depolymerizing factor, ADF). Phosphorylation of cofilin serine-3 inactivates the actin severing ability promoting F-actin polymerization, stress fibre formation and focal adhesion formation. LIM kinases also undergo nuclear/cytoplasmic shuttling and several nuclear substrates are suggested, including CREB (8). During pro-metaphase and metaphase LIMKs are maximally phosphorylated and are localized to spindle poles (9). A reduction of LIMK1 induces G2/M cell cycle block suggesting an important role for these kinases in mitosis and cytokinesis.

Several proteins have been shown to interact and regulate the LIM kinases, including Hsp90, p57KIP2, RNF6 and Par-3. Notably, the LATS (large tumour suppressor) kinase has been shown to inhibit LIMK1 (10). A direct interaction is also suggested between the LIMK kinase domain and the N-terminal LIM domains (11). This appears to fulfill a regulatory function as N-terminal deletion is sufficient to activate LIMK kinase activity. Both the LIM-LIM and PDZ domains may also mediate additional protein interactions. For example, BMP-dependent dendritogenesis requires an interaction between the LIM-LIM domains and the C-terminal tail region of the BMP receptor BMPR2 (12). This interaction is lost in some cases of pulmonary arterial hypertension (PAH) where the BMPR2 tail is deleted by non-sense mutation. Conversely, loss of FMRP in fragile X syndrome stabilises the mRNA of BMPR2 and consequently drives excessive activation of the BMPR2-LIMK1-cofilin pathway leading to stable actin and abnormal synapse and dendritic spine development. These morphological abnormalities could be rescued by BMPR2 knockdown or by postnatal pharmacological inhibition of LIMK1 activity (13).

Here, we solved the crystal structure of the LIMK1 kinase domain in complex with bound ATPγS and substrate CFL1 (Ser3Cys mutant). The structure confirms a novel kinase-substrate interface that allows globular cofilin to be phosphorylated.

Materials and Methods

XX02LIMK1A-d001 : LIMK1 + CFL1 + ATP-Î³-S; Adenosine 5'-[gamma-thio]triphosphate tetralithium salt: human LIM kinase 1 (LIMK1) in complex with CFL1 and ATP-Î³-S

PDB Code: 5L6W

Material and Methods

SGC target ID: LIMK1

Entry Clone Source: TKC

Entry Clone DNA Sequence:

TACTTCCAATCCATGCCACACCGCATCTTCCGGCCGTCGGACCTCATCCACGGGGAGGTGCTGGGCAAGGGCTGCTTC
GGCCAGGCTATCAAGGTGACACACCGTGAGACAGGTGAGGTGATGGTGATGAAGGAGCTGATCCGGTTCGACGAGGAG
ACCCAGAGGACGTTCCTCAAGGAGGTGAAGGTCATGCGATGCCTGGAACACCCCAACGTGCTCAAGTTCATCGGGGTG
CTCTACAAGGACAAGAGGCTCAACTTCATCACTGAGTACATCAAGGGCGGCACGCTCCGGGGCATCATCAAGAGCATG
GACAGCCAGTACCCATGGAGCCAGAGAGTGAGCTTTGCCAAGGACATCGCATCAGGGATGGCCTACCTCCACTCCATG
AACATCATCCACCGAGACCTCAACTCCCACAACTGCCTGGTCCGCGAGAACAAGAATGTGGTGGTGGCTGACTTCGGG
CTGGCGCGTCTCATGGTGGACGAGAAGACTCAGCCTGAGGGCCTGCGGAGCCTCAAGAAGCCAGACCGCAAGAAGCGC
TACACCGTGGTGGGCAACCCCTACTGGATGGCACCTGAGATGATCAACGGCCGCAGCTATGATGAGAAGGTGGATGTG
TTCTCCTTTGGGATCGTCCTGTGCGAGATCATCGGGCGGGTGAACGCAGACCCTGACTACCTGCCCCGCACCATGGAC
TTTGGCCTCAACGTGCGAGGATTCCTGGACCGCTACTGCCCCCCAAACTGCCCCCCGAGCTTCTTCCCCATCACCGTG
CGCTGTTGCGATCTGGACCCCGAGAAGAGGCCATCCTTTGTGAAGCTGGAACACTGGCTGGAGACCCTCCGCATGCAC
CTGGCCGGCCACCTGCCACTGGGCCCACAGCTGGAGCAGCTGGACAGAGGTTTCTGGGAGACCTACCGGCGCGGCGAG
AGCTGACAGTAAAGGTGGATA

Vector: pFB-LIC-Bse

Tag: N-terminal His6-TEV

SCG Construct ID : LIMK1A-c056

Construct Protein Sequence:
MGHHHHHHSSGVDLGTENLYFQSMPH
RIFRPSDLIHGEVLGKGCFGQAIKVT
HRETGEVMVMKELIRFDEETQRTFLK
EVKVMRCLEHPNVLKFIGVLYKDKRL
NFITEYIKGGTLRGIIKSMDSQYPWS
QRVSFAKDIASGMAYLHSMNIIHRDL
NSHNCLVRENKNVVVADFGLARLMVD
EKTQPEGLRSLKKPDRKKRYTVVGNP
YWMAPEMINGRSYDEKVDVFSFGIVL
CEIIGRVNADPDYLPRTMDFGLNVRG
FLDRYCPPNCPPSFFPITVRCCDLDP
EKRPSFVKLEHWLETLRMHLAGHLPL
GPQLEQLDRGFWETYRRGES
Residues marked in red are cleaved by TEV protease

Host: Insect cells (Sf9)

Expression & Harvest:

Exponentially growing Sf9 cells (2x 106 cells/mL) were infected with high titre baculovirus stock (1:65) and incubated in shaker flasks (60 hours, 90 rpm, 27°C). Following this, the cell suspensions were centrifuged (15 minutes, 800x g, 4°C), and the cell pellets resuspended in PBS. After another centrifugation (15 minutes, 800x g, 4°C), the cell pellets were stored at -80°C.

Purification:

Lysis Buffer:50 mM Hepes pH 7.4, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP.
Binding Buffer:50 mM Hepes pH 7.4, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol, 0.5 mM TCEP.
Wash Buffer 1:As Binding Buffer except 1 M NaCl and 40 mM imidazole.
Wash Buffer 2:As Binding Buffer except 60 mM imidazole.
Elution Buffer:As Binding Buffer except 300 mM imidazole.
The cell pellet was thawed, resuspended in 100 mL lysis buffer and sonicated (2 minutes, amplitude 35%, on ice). The cell lysate was cleared by centrifugation (60 minutes, 36,000x g, 4°C). The supernatant was combined with 7.5 mL Ni Sepharose and loaded onto a gravity flow column. The column was washed with 100 mL of Binding Buffer and 50 mL each of Wash Buffer 1 and 2. 25 ml of Elute Buffer was passed through to elute the protein. Fractions containing protein were pooled, combined with recombinant TEV protease (mass ratio 1:25) and incubated with rotation (over night, 4°C). Following this, 7.5 mL Ni Sepharose was added, and the suspension was loaded onto a gravity flow column. The flowthrough was collected and concentrated to 5 mL. Finally, the protein was polished by gel filtration using an AKTAxpress system with an S200 16/600 column and GF buffer (20 mM HEPES/NaOH pH 7.4, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP). Fractions containing protein were pooled, concentrated and stored at -80°C.

SGC target ID: CFL1

Entry Clone Source: MGC

Entry Clone DNA Sequence :
CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGA
GAACCTGTACTTCCAATCCATGGCCTGCGGTGTGGCTGTCTCTGATGGTG
TCATCAAGGTGTTCAACGACATGAAGGTGCGTAAGTCTTCAACGCCAGAG
GAGGTGAAGAAGCGCAAGAAGGCGGTGCTCTTCTGCCTGAGTGAGGACAA
GAAGAACATCATCCTGGAGGAGGGCAAGGAGATCCTGGTGGGCGATGTGG
GCCAGACTGTCGACGATCCCTACGCCACCTTTGTCAAGATGCTGCCAGAT
AAGGACTGCCGCTATGCCCTCTATGATGCAACCTATGAGACCAAGGAGAG
CAAGAAGGAGGATCTGGTGTTTATCTTCTGGGCCCCCGAGTCTGCGCCCC
TTAAGAGCAAAATGATTTATGCCAGCTCCAAGGACGCCATCAAGAAGAAG
CTGACAGGGATCAAGCATGAATTGCAAGCAAACTGCTACGAGGAGGTCAA
GGACCGCTGCACCCTGGCAGAGAAGCTGGGGGGCAGTGCCGTCATCTCCC
TGGAGGGCAAGCCTTTGTGACAGTAAAGGTGGATACGGATCCGAA

SCG Construct ID :CFL1A-c002

Construct Protein Sequence :
MHHHHHHSSGVDLGTENLYFQSMACGVAVSDGVIKVFNDMKVRKSSTPEEV
KKRKKAVLFCLSEDKKNIILEEGKEILVGDVGQTVDDPYATFVKMLPDKDC
RYALYDATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKKLTGI
KHELQANCYEEVKDRCTLAEKLGGSAVISLEGKPL
This is a variant protein S3C. Cysteine instead of Serine. Residues marked in red are cleaved by TEV protease.

Vector: pNIC28-Bsa4

Tag:N-terminal His6-TEV

Host:BL21

Expression & Harvest Transformation:The CFL1 construct DNA was transformed into homemade competent cells of the expression strain by a standard heat shock procedure. Colonies were used to inoculate 50 mL of LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol, which was placed in a 37°C shaker overnight. The next day 4x 10 mL of this starter culture was used to inoculate 4x 1L of LB media containing 37.5 µg/mL kanamycin in 2L baffled shaker flasks. When the OD600 was approximately 0.45, the temperature was reduced to 20°C and when the OD600 was approximately 0.6 the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight. Cells were spun at 5000rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -80°C.

Purification:

Lysis Buffer:50 mM Hepes pH 7.4, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP.
Binding Buffer:50 mM Hepes pH 7.4, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol, 0.5 mM TCEP.
Wash Buffer 1:As Binding Buffer except 1 M NaCl and 40 mM imidazole.
Wash Buffer 2:As Binding Buffer except 60 mM imidazole.
Elution Buffer:As Binding Buffer except 300 mM imidazole.
The cell pellet was thawed, resuspended in lysis buffer and sonicated (12 minutes, amplitude 35%, on ice). The cell lysate was cleared by centrifugation (60 minutes, 36,000x g, 4°C). The supernatant was combined with 7.5 mL Ni Sepharose and loaded onto a gravity flow column. The column was washed with 100 mL of Binding Buffer and 50 mL each of Wash Buffer 1 and 2. 25 ml of Elute Buffer was passed through to elute the protein. Fractions containing protein were pooled, combined with recombinant TEV protease (mass ratio 1:25) and incubated with rotation (over night, 4°C). Following this, 7.5 mL Ni Sepharose was added, and the suspension was loaded onto a gravity flow column. The flowthrough was collected and concentrated to 5 mL. Finally, the protein was polished by gel filtration using an AKTAxpress system with an S200 16/600 column and GF buffer (20 mM HEPES/NaOH pH 7.4, 500 mM NaCL, 5% Glycerol, 0.5 mM TCEP). Fractions containing protein were pooled, concentrated and stored at -80°C.

LIMK1 and CFL1
The LIMK1 and CFL1 protein were mixed in to 1:1 ratio and incubated for 2 hours on ice. The protein was polished by gel filtration using an AKTAxpress system with an S200 16/600 column and GF buffer (20 mM HEPES/NaOH pH 7.4, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP).

Crystallisation
ATP-Î³-S was added to the protein final concentration of 1 mM and was concentrated to 10 mg/ml. Crystals grew from a 1:2 ratio of protein solution (10 mg/mL) and precipitant solution (0.2M potassium chloride - 35% pentaerythritol propoxylate 5/4 - 0.1M HEPES pH 7.5), using the vapour diffusion method.

Data Collection: Resolution: 2.53Å
X-ray source:Crystals were cryo-protected by equilibration into precipitant solution containing 20% ethylene glycol and flash frozen in liquid nitrogen. Data was collected at Diamond, beamline I02.

References

1. Scott RW, Olson MF 2007 LIM kinases: function, regulation and association with human disease. Journal of molecular medicine (Berlin, Germany) 85:555-568

2. Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M, Lu WY, MacDonald JF, Wang JY, Falls DL, Jia Z 2002 Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 35:121-133

3. Takahashi H, Koshimizu U, Miyazaki J, Nakamura T 2002 Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene. Developmental biology 241:259-272

4. Frangiskakis JM, Ewart AK, Morris CA, Mervis CB, Bertrand J, Robinson BF, Klein BP, Ensing GJ, Everett LA, Green ED, Proschel C, Gutowski NJ, Noble M, Atkinson DL, Odelberg SJ, Keating MT 1996 LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell 86:59-69

5. Tassabehji M, Metcalfe K, Fergusson WD, Carette MJ, Dore JK, Donnai D, Read AP, Proschel C, Gutowski NJ, Mao X, Sheer D 1996 LIM-kinase deleted in Williams syndrome. Nature genetics 13:272-273

6. Yoshioka K, Foletta V, Bernard O, Itoh K 2003 A role for LIM kinase in cancer invasion. Proceedings of the National Academy of Sciences of the United States of America 100:7247-7252

7. Bernard O 2007 Lim kinases, regulators of actin dynamics. The international journal of biochemistry & cell biology 39:1071-1076

8. Yang EJ, Yoon JH, Min DS, Chung KC 2004 LIM kinase 1 activates cAMP-responsive element-binding protein during the neuronal differentiation of immortalized hippocampal progenitor cells. The Journal of biological chemistry 279:8903-8910

9. Kaji N, Muramoto A, Mizuno K 2008 LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning. The Journal of biological chemistry 283:4983-4992

10. Yang X, Yu K, Hao Y, Li DM, Stewart R, Insogna KL, Xu T 2004 LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1. Nature cell biology 6:609-617

11. Foletta VC, Lim MA, Soosairajah J, Kelly AP, Stanley EG, Shannon M, He W, Das S, Massague J, Bernard O 2003 Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. The Journal of cell biology 162:1089-1098

12. Lee-Hoeflich ST, Causing CG, Podkowa M, Zhao X, Wrana JL, Attisano L 2004 Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. The EMBO journal 23:4792-4801

13. Kashima R, Roy S, Ascano M, Martinez-Cerdeno V, Ariza-Torres J, Kim S, Louie J, Lu Y, Leyton P, Bloch KD, Kornberg TB, Hagerman PJ, Hagerman R, Lagna G, Hata A. 2016. Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome. Sci Signal. 9(431):ra58.