Download Crystal structure of the nuclear factor hCINAP in complex with ADP

Crystal structure of the nuclear factor hCINAP in complex with ADP refined to 1.8 Å resolution :
Insights into the function of hCINAP.
1,*
2
2
1
1
1
3
1
Spyros E. Zographos , Anna Malekkou , Niovi Santama , Demetres Papageorgiou , Eliada Lazoura , Demetres D. Leonidas , Angus I. Lamond and N. G. Oikonomakos
1
2
Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48, Vas. Constantinou Ave. Athens 11635, Greece. Department of
3
Biological Sciences, University of Cyprus and Cyprus Institute of Neurology and Genetics, PO Box 20537, 1678 Nicosia, Cyprus Division of Gene Regulation and
*
Expression, University of Dundee, MSI/WTB Complex, Dundee DD1 5EH, Scotland, UK. Corresponding author, tel.: +30-2107273895, [email protected]
Indroduction.
Human Coilin Interacting Nuclear ATPase Protein (hCINAP) is a nuclear factor composed of 172 amino acids which includes
a phosphate binding loop (P-loop). Both the nuclear localization and identified protein partners of hCINAP suggest the
possible involvement of this protein in transcription and/or RNA processing. Recently, we reported that hCINAP can interact
with coilin, a marker protein of Cajal bodies which are well-known to be involved in nuclear splicing. Additionally, using an
ATPase assay we demonstrated that hCINAP possesses significant ATPase activity and also shows a high affinity for ATP [1].
Herein, we report the structure of hCINAP in complex with ADP, determined at 1.8 Å resolution and refined to an R-factor of
0.179 (Rfree = 0.205). Binding studies have revealed the location of the catalytic site of hCINAP and have provided insight into
which amino acids are involved in nucleotide recognition. Based on this structural analysis, site-directed mutagenesis studies
have been employed to further evaluate the function and precise biological role of the protein in the mammalian nucleus.
Figure 1. 2FO-FC electron density map showing ADP bound at the catalytic site. The contour level corresponds to approximately 1ó
deviation of the map. Water molecules are represented as red spheres while P-loop residues are shown in yellow.
Results and Discussion.
Inspection of the difference electron density maps of the hCINAP-ADP complex clearly shows a strong Fo-Fc accompanied by
a 2Fo-Fc electron density which can accommodate one ADP molecule (Figure 1). ADP binds in a groove located near the
protein surface with the adeninine ring occupying the entrance of the catalytic site and the phosphate groups orientated toward
the P-Loop region.
The crystal structure further shows that upon binding to hCINAP, ADP forms numerous contacts, both polar and non-polar,
with P-Loop residues (a.a. 10-17), while also making three additional hydrogen bonds with the side chain of a conserved Arg
residue (Figure 2). In total, ADP forms 24 hydrogen bonds and 122 van der Waals contacts, displacing the 7 water molecules
present in the native form (1RKB, [2]). Negligible overall conformational changes are observed in the tertiary structure of
hCINAP, associated with the co-crystallisation of the protein with ADP. The root mean square (rms) difference in Cá atoms
with respect to native hCINAP is 0.576 Å. Within the catalytic site there are no significant differences in amino acid side chain
position except for a shift, as high as 1.7 Å, in the side chain atoms of Arg109, which is associated with conformational changes
to optimize the contact with the â-phosphate group. Furthermore, the structural analysis has revealed that a DXXG motif (âmotif, a.a. 77-80), which is highly conserved in the GTPase superfamily, contributes to the nucleotide binding site, thus
suggesting possible dual ATPase/GTPase enzymatic activity. Indeed, the His79 ring shifts by 2.5 Å towards ADP in order to
coordinate a magnesium ion via water molecules (data not shown). The major conformational change upon binding of ADP
(rms difference in Ca atoms with respect to native is 0.8 to 2.9 Å) occurs in the surface loop, which comprises of amino acids
45-55 and adopts a â-sheet-turn-â-sheet conformation (Figure 3). However, this loop is separate from the catalytic site and
there is no rational explanation for this conformational change.
The crystal structure of the native protein has previously been reported [2] where the authors proposed that hCINAP be
designated an adenylate kinase (AK6) based on its structural similarities with other members of the AK family. However, it
was assigned to a distinct subfamily due to its unusually broad substrate specificity and distinct structural features compared
with other known human adenylate kinase classes, AK1-5. Although, in our view, the assignment of hCINAP as a bona fide
adenylate kinase remains to be validated, our present data does confirm its status as an ATPase. We have shown that
recombinant hCINAP exhibits high apparent ATP affinity and ATPase activity. The Km value for ATP was calculated to be 75.3
± 5.0 µM with a Vmax value of 1.27 ± 0.2 µmole ADP formed min-1. mg-1. (Figure 4). Structural analysis did not propose any
putative binding site for a second ADP molecule as would be expected for an adenylate kinase. Furthermore, the possible
binding position of phosphate groups, as indicated by the identified sulphate binding sites, are located on the protein surface
and are distant from the catalytic site. Based on these findings we have performed mutational studies of hCINAP, which have
shown that single point mutations of specific residues involved in the ADP binding alter the cellular localization of hCINAP,
while double mutations lead to increased levels of apoptotic death (data not shown). To further investigate the catalytic
mechanism of nucleotide triphosphate (NTP) hydrolysis, comparative kinetic and crystallographic studies of native and
mutant hCINAP in complexes with a number of NTPs, dNTPs and non hydrolysable NTP analogues are currently underway.
Elucidating the biological function of hCINAP is of great interest, as it may be a key molecule linking the basal transcription
machinery and aspects of pre-mRNA metabolism. Detailed understanding of hCINAP function and its interaction with protein
partners and small molecules will be instrumental in putting the structure/function relationship of this new protein into
biological context.
Materials and Methods
hCINAP from human skeletal muscle was cloned, expressed and purified as previously described [1]. ATP hydrolysis activity
of hCINAP was measured by an assay [3] in which ADP production is coupled to the â-NADH oxidation by pyruvate kinase
(PK) and L-Lactic dehydrogenase (LDH), with minor modifications [1].
hCINAP crystals, with an average size of 0.3 - 0.5 mm, were obtained using the sitting drop vapour diffusion technique in a
buffer comprising of 15 mg/ml enzyme, 0.1 M Hepes pH 7.5, 1.5 M Li2SO4, 0.2 M NaCl and 0.5 mM DTT. hCINAP co-crystals
grew within a week in the presence of 2 mM ADP and 25 mM Mg2+ at 20 ºC (Image 1). Prior to data collection, the crystals were
transferred to fresh buffer solution containing 25% v/v glycerol for 5 - 15 sec prior to flash freezing (100 K). Diffraction data
were collected from a single crystal on the beamline PX 10.1 at SRS, Daresbury Laboratory (ë = 1.117 Å) using a MAR CCD
detector. The crystal to image plate distance was 150 mm and gave a maximum resolution of 1.8 Å at the edge of the detector.
Data frames of 0.8° oscillation angle were collected over a total angular range of 80°. Integration and data reduction were
performed with the programs DENZO and SCALEPACK [4] and intensities were transformed to amplitudes by the program
TRUNCATE [5]. The crystals were isomorphous with the native form and belonged to the P61 space group (a=b= 98.848, c=
57.468) with one monomer per asymmetric unit. Phases were obtained using the structure of free hCINAP (1RKB, [2]) as
starting model. Alternate cycles of manual building with the program Coot [6] and refinement using the maximum likelihood
target function as implemented in the program REFMAC [7] improved the model phases. At this stage water molecules were
added to unidentified Fo-Fc map peaks greater than 1.0ó by using the “water find” module of the program Coot [6]. After an
additional cycle of refinement and manual building, the ADP molecule was included in subsequent refinement cycles. The ADP
molecule was retrieved from the small molecule REFMAC library and fitted into the electron density using the program Coot.
The final model generated by a round of TLS (Translation/Libration/Screw) refinement within the program REFMAC [7] using
TLS groups for the protein generated by the TLSMD web server [8]. Details of data processing and refinement statistics are
summarized in Table 1. The program PROCHECK [9] was used to assess the quality of the final structure. Analysis of the
Ramachandran (uw) plot showed that all residues lie in the allowed regions. Figures were prepared and rendered with the
program PyMol [10].
Table 1. Crystallographic statistics.
Structure
hCINAP-ADP
Space group
P61
Cell dimensions (Å), a,b,c 98.85, 98.85, 57.47
Resolution (Å)
30-1.79
Outermost shell (Å)
1.82-1.79
Reflections measured
152,209
Unique reflections
32,104
Rsymm (Outermost shell)
0.044 (0.447)
Completeness %
99.7 (99.4)
<I/óI> (Outermost shell) 17.9 (4.1)
Rcryst % (Outermost shell)
0.179 (0.226)
Rfree % (Outermost shell)
0.205 (0.235)
No of protein atoms
1,510
No of solvent molecules 217
Rms deviation from ideality
in bond lengths (Å)
0.009
in angles (º)
1.24
Average B factor
Protein atoms (Å2)
29.99
Solvent molecules (Å2)
52.59
Image 1. hCINAP hexagonal crystals grew in the presence of 2
2+
mM ADP and 25 mM Mg by sitting drop technique.
Figure 2 Ribbon representation of hCINAP catalytic site showing the hydrogen bond interactions (denoted by dashed lines) of ADP
with the protein. Water molecules are represented as red spheres, while P-loop residues are shown in yellow.
Figure 4. Plot of initial ATP hydrolysis velocities vs. various ATP concentrations (0.05 to 1 mM).
Inset: Double reciprocal plot.
Figure 3. Structure superimposition of hCINAP-ADP complex (green) onto the free hCINAP (1RKB) (purple). N- and C-termini
are represented as blue and red sphere, respectively. Seven-residue cloning artifact at protein N-termini is shown in white.
Acknowledgements.
This work was supported by the Greek GSRT through ENTER (2004-2006) 01EP115 to SEZ and NGO, the Research
Promotion Foundation (Cyprus) through RPF'S FP 2006, YGEIA/o506/05 to NS and EU through a Marie Curie European
Reintegration Grant, MERG-CT-2004-006358 (2004-2006) to SEZ.
We thank Ms. Kyriaki Dossi for kindly providing technical assistance on protein purification and X-ray data collection. We
also thank Ms. Christina Drakou for assistance with data processing and structure refinement.
References
1. Santama, N., Ogg, S. C., Malekkou, A., Zographos, S. E., Weis, K. & Lamond, A. I. (2005). Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID32 locus.
J Biol Chem 280, 36429-41
2. Ren, H., Wang, L., Bennett, M., Liang, Y., Zheng, X., Lu, F., Li, L., Nan, J., Luo, M., Eriksson, S., Zhang, C. & Su, X. D. (2005). The crystal structure of human adenylate kinase 6: An adenylate kinase localized to the cell nucleus. Proc Natl Acad Sci
U S A 102, 303-8.
3. Skamnaki, V. T., Owen, D. J., Noble, M. E., Lowe, E. D., Lowe, G., Oikonomakos, N. G. & Johnson, L. N. (1999). Catalytic mechanism of phosphorylase kinase probed by mutational studies. Biochemistry 38, 14718-30.
4. Otwinowski, Z. & Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Macromolecular Crystallography, Pt A 276, 307-326.
5. French, S. & Wilson, K. (1978). Treatment of Negative Intensity Observations. Acta Crystallographica Section A 34, 517-525.
6. Emsley, P. & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography 60, 2126-2132.
7. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D-Biological Crystallography 53, 240-255.
8. Painter, J. & Merritt, E. A. (2006). TLSMD web server for the generation of multi-group TLS models. Journal of Applied Crystallography 39, 109-111.
9. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. (1993). Procheck - a Program to Check the Stereochemical Quality of Protein Structures. Journal of Applied Crystallography 26, 283-291.
10.DeLano, W. L. (2002). The PyMOL Molecular Graphics System, DeLano Scientific, Palo Alto, CA, USA.