by Quan Hao*
*on behalf of team members: Qun Liu, Irina Kriksunov, Qingqiu Huang, Xinguo Hong, Jun Fan, David Schuller, Qiuyue Yang & Yeyun Zhou
Overall goal: our goal is to develop computational and experimental methods that drive structural studies of biologically important proteins. This report starts with an overview that summarizes our research accomplishments since 2001, as well as the future research directions. In the remaining pages, each project is addressed in more depth.
Development of new macromolecular phasing methods.
Biological problem. X-ray crystallography is foremost among the methods used to determine the atomic-scale structure of macromolecules. However, the inversion from measured x-ray diffraction intensities to atomic structure is not as straightforward as one might think: The intensity relates to the amplitude of a scattered wave, but not to its phase relative to the other scattered waves. Without those phases, one simply does not know how to add all scattered waves together to retrieve the original structure, especially when that structure is a macromolecule such as a protein or virus. That difficulty is called the phase problem of x-ray crystallography.
Our approach. Most novel protein structures have been
solved by Multiple-wavelength Anomalous Dispersion (MAD) technique. The MAD
technique involves measuring the diffraction intensities at multiple x-ray
wavelengths near an absorption edge of an incorporated atom such as Se. Since
most protein crystals are easily damaged by radiation, it is desirable to
reduce the number of data sets required to solve a structure. In the cases
where the absorption edge of the anomalous scatterer (for example, sulfur) is
outside the accessible wavelength range, MAD phasing is not even a possibility.
In collaboration with Prof. Hai-fu Fan of the
An emerging method. Envelope phasing is a promising technique that does not require the presence of incorporated atoms; it instead utilizes the low-resolution shape, or envelope, of the protein. The envelope may be determined by small-angle x-ray scattering of a protein in solution, electron-microscope images of isolated molecules. To utilize the envelope information, a crystallographer must determine the known envelope's orientation and position in a crystallographic unit cell. We have developed a method to perform a simultaneous 6D search on orientation and translation to find the best match between structure factors determined by experiment and those calculated from a presumed location of the envelope in the unit cell. The method has been successfully tested on several biological systems.
Continuing development. The techniques involved depend heavily on mathematics and statistics, and are formulated into computer programs. There is a continuing need to develop these programs as techniques change and X-ray equipment and computers become more powerful and sophisticated. We have written four phasing programs based on the above methods and these programs are used in the world-wide diffraction community as part of the open CCP4 software suite.
The Laue method allows large number of reflections to be collected on a single image without oscillating the crystal. A new approach to Laue diffraction was taken in an experiment at CHESS G-1 station using multilayer x-ray optics instead of the silicon monochromator found at the usual crystallography stations. The number of frames required for a useful dataset is comparable to that for a monochromatic oscillation experiment, but the exposure time per frame is considerably less. The narrow-bandpass Laue technique shows promise for rapid data collection, or for data collection on a bending magnet line with exposure times typical for an insertion device line. This technique may also be appropriate for the proposed data collection experiments from ‘a shower’ of randomly orientated micro-crystals.
Crystallography is the major technique for elucidating structure-function
relationships. The results can
provide insights into how biological systems work and may find wide reaching
applications in pharmaceutical design. Specific themes in my group include
signaling proteins, enzyme substrate structures and catalysis. One project,
that has excited media interest, is on a multi-functional enzyme, CD38. Human
CD38 is physiologically recruited for the synthesis of several calcium
messengers. We also determined the complex structures of the enzyme with its
substrate, and with products. In addition to its enzymatic function, human
CD38 is a cell surface transmembrane receptor and is directly involved in a
number of diseases such as B cell chronic lymphocytic leukemia (B-CLL), AIDS,
Diabetes, asthma, arthritis, and inflammation. As some of these diseases
(diabetes, asthma, and inflammation) have been shown to be related to the
enzymatic function of human CD38, these complex structures
may provide significant advance for the structure-based design of human
CD38 inhibitors that could be drug candidates for these diseases.
Another project area being developed involves structural studies of reaction intermediates. It is generally accepted that the catalytic ability of an enzyme relies greatly on the structure of the intermediate, a transient state occurs during the transition from reaction substrate to product. By using X-ray crystallographic tools, we captured and determined the structures of reaction intermediates from two different enzymes. We hope to extend the technique to study CD38 and other interesting biological systems.
Detailed project description
1. Direct phasing of Single-wavelength Anomalous
Diffraction (SAD) data.
(In collaboration with Professor Fan, Hai-fu's Group at the
Most novel protein structures have been solved by multiple wavelength
anomalous dispersion (MAD) technique. The MAD technique involves measuring the
diffraction intensities at multiple x-ray wavelengths near an absorption edge
of an incorporated atom such as Se. Since most protein crystals are easily
damaged by radiation, it is desirable to reduce the number of data sets
required to solve a structure (1).
Together with the Fan group, we have developed a direct-methods procedure (based on phase relationships to
resolve the phase ambiguity arising from single wavelength anomalous dispersion
(SAD) data. The method has been implemented in the computer program OASIS
(2) which is now a fully
supported program in the CCP4
Suite. We have recently solved the crystal structures of atratoxin (3), cysteine dioxygenase (4), and polyamine oxidase (5) with this method. We have also
developed a program SAPI (6) to find heavy atom sites using
SAD data and a program ABS (7)
to determine the absolute configuration of the heavy atom substructure. Our
advance in phasing methods also helps others to solve interesting but difficult
structures. For example, with our input, Yigong Shi (
Future work. The class of proteins and macromolecules which can be studied via X-ray diffraction is expanding, and the methodology must respond to these new challenges. The current focus of this project is to extend the boundaries of structural solutions that are now possible. Bijvoet amplitude ratio (<|ΔF|>/<F>) of 0.6 % was generally regarded as the limit for SAD phasing. We have recently successfully used a single 0.31 occupied krypton site in a porcine pancreas elastase (26 kDa) molecule (Bijvoet amplitude ratio of 0.53 %) for SAD phasing (9).
2. Molecular Envelope determination and ab initio phasing
We have developed a method for locating a molecular envelope (determined by the small angle scattering technique, EM or other methods) in crystallographic unit cell. The low-resolution envelope can be used as a starting model for phase extension by the maximum entropy and density modification method. The crystal structure can therefore be determined without the requirement of incorporating heavy atoms in the protein. In a test case, the low resolution molecular envelope of nitrite reductase (NiR) determined from solution X-ray scattering data was located in the crystallographic unit cell by a molecular search method. A computer program, FSEARCH (10,11) based on this method has been included in the CCP4 Suite. The FSEARCH program has also been used to find a molecular replacement solution with data from the 420 kDa lobster clottable protein crystals; the search model was a 17 Å resolution structure determined by single particle EM (12).
Future work. The low-resolution phases calculated from the correctly positioned molecular envelope need to be extended to higher (crystallographic) resolution. The phase extension is a very challenging problem and requires substantial amount of effort in developing new methods. The standard density modification methods such as solvent flattening, histogram matching, non-crystallographic averaging and maximum entropy are known to be most effective for phase extension in the resolution range 5 Å or higher. To bridge the gap between the envelope resolution (usually in the range of 10 to 20 Å) and 5 Å, we have proposed new methods such as the Genetic Algorithm (GA) and the Iterative Projections method (in collaboration with Veit Elser, Cornell Physics Dept.).
3. Laue Diffraction & Wide-Bandpass Optics.
We are particularly interested in the energy-overlapping problem that has limited Laue data completeness and therefore affected the quality of electron density map. We have proposed and successfully implemented methods based on direct methods and the maximum entropy technique (13). In particular, the maximum entropy method does not require data redundancy and every multiple diffraction spot can be deconvoluted to satisfactory quality. Indeed, the map connectivity has been improved significantly by inclusion of these deconvoluted reflections. I have also devoted a great deal of effort in developing the Daresbury Laue Suite. The Laue diffraction data processed by the Suite were of similar quality to the monochromatic data. The Laue method offers great potential in time-resolved studies.
A new approach to Laue diffraction was taken in an experiment at CHESS G-1 station using multilayer x-ray optics instead of the silicon monochromator found at the usual crystallography stations. A total of 72 1-second Laue exposures from a lysozyme crystal were taken at 1 degree intervals. They were processed with a combination of monochromatic and Laue software and used for structure determination by the molecular replacement technique. The crystallographic R after structure refinement was a very satisfactory 20%. The x-ray spectrum from the pair of multilayers showed good intensity over a wavelength range of about 0.975-0.995 A (1.7% bandwidth), as compared with a typical Laue range of 0.7-1.7 A. With this spectrum, diffraction data contain essentially no harmonic overlaps, at the expense of relatively few reflections per exposure. The number of frames required for a useful dataset is comparable to that for a monochromatic oscillation experiment, but the exposure time per frame is considerably less.
Future work. The narrow-bandpass Laue technique shows promise for rapid data collection, or for data collection on a bending magnet line with exposure times typical for an insertion device line. This technique may also be appropriate for the proposed data collection experiments from ‘a shower’ of randomly orientated micro-crystals (Gruner, Bilderback, ERL presentations).
4. Crystal structure of human CD38
Figure 4. This artistic rendering of the molecular structure of human CD38 appears on the cover of the 2005/9 issue of the journal Structure (credit: Qun Liu).
CD38 is a novel multifunctional ectoenzyme widely expressed in
cells and tissues especially in leukocytes. CD38 also functions in cell
adhesion, signal transduction and calcium signaling. In collaboration with HC
Lee (U. Minnesota, now at
The findings also may offer insights into mechanisms involved in certain diseases, ranging from leukemia to diabetes and HIV-AIDS. As one example, researchers have shown that CD38 interrupts an interaction between the AIDS virus and its point of entry into cells -- a protein receptor called CD4. By looking at CD38's 3-D structure, we identified a peptide that may play a role in interrupting the interface between CD4 and HIV-AIDS.
The findings, published in the journal Structure (14), mark an essential step toward designing drugs that could inhibit processes related to certain diseases. Knowing the protein's structure also opens the door to understanding CD38's many functions related to key biological processes about which researchers know very little. For example, the molecular mechanism of how a cell mediates calcium release is largely unknown. So this is a very fundamental question for biologists. It turns out that CD38 helps produce at least two calcium messenger molecules, each of which then opens channels for the release of calcium from specific stores, or reservoirs, within cell organelles.
Future work. The CD38 project has been funded by an
NIH RO1 grant with two performance sites:
5. Structural studies of reaction intermediates
Understanding of the atomic movements involved in an enzymatic reaction needs structural information on the active and inactive native enzyme molecules, and on the enzyme-substrate, enzyme-intermediate and enzyme-product(s) complexes. Two enzymes have been studied: ADP-ribosyl cyclase (15) and Aspergillus fumigatus Phytase (16,17). We have determined to 2.0 Å resolution the structure of Aplysia cyclase in a covalent complex with ribose-5-phosphate and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In the 1.5Å resolution crystal structure of Aspergillus fumigatus Phytase, the catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family.
Future work. The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD+) has been proposed to go through an oxocarbenium ion-like transition state. Due to the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD+ to synthesize two calcium mobilizing molecules. We plan to determine high resolution crystal structures of the enzyme complexed with the intermediate, with substrates and with products. The structural analysis of these complexes would provide insights into the mechanisms of nicotinamide cleavage, intermediate stabilization and products generation.
6. Other work
We enjoy collaborating with colleagues both inside and outside of Cornell to work on interesting technical and biological problems. Below is a list of projects that we have accomplished in the last few years:
· Blocking Effect and Crystal Structure of Natrin Toxin, a Cysteine-Rich Secretory Protein from Naja atra Venom that Targets the BKCa Channel (23).
· Crystal Structure of the Cysteine-rich Secretory Protein Stecrisp Reveals That the Cysteine-rich Domain Has a K+ Channel Inhibitor-like Fold (24,25).
· Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom (26).
· Crystallographic data collection using multilayer optics (27,28).
· S-SWAT (softer single-wavelength anomalous technique): potential in high-throughput protein crystallography (29).
1. Q. Shen, Q. Hao, and S. M. Gruner (2006) Physics Today, March 2006, 46-52. Macromolecular Phasing (Invited review).
2. Q. Hao, Y.X. Gu, C.D. Zheng, & H.F. Fan (2000) J. Appl. Cryst. 33, 980-981. OASIS: A Program for Breaking Phase Ambiguity in OAS or SIR.
3. X. Lou, Q. Liu, X. Tu, J. Wang, M. Teng, L.W. Niu, D.J. Schuller, Q. Huang, Q. Hao (2004) J. Biol. Chem., vol 279, 39094-39104. “The Atomic Resolution Crystal Structure of Atratoxin Determined by SAD Phasing”
4. C. R. Simmons, Q. Hao and M. H. Stipanuk (2005) Acta Cryst. F61, 1013-1016. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.
5. Q. Huang, Q. Liu and Q. Hao (2005). J. Mol. Biol. Vol 348, 951-959. “Crystal Structures of Fms1 and its Complex with Spermine Reveal Substrate Specificity.”
6. Hao Q., Gu
7. Hao Q. (2004) J. Appl. Cryst. 37, 498-499. “ABS: a program to determine absolute configuration and evaluate anomalous scatterer substructure”.
8. N. Yan, J. Chai, E. S. Lee, L. Gu, Q. Liu, J. He, J.-W. Wu, D. Kokel, H. Li, Q. Hao, D. Xue and Y. Shi (2005) Nature, 437, 831-837. Structure of the CED-4−CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans.
9. C.U.Kim, Q. Hao & S.M. Gruner (2006), Acta Cryst. D62, in press
10. Liu Q., Weaver A.J., Xiang T., Thiel D.J. and Hao Q. (2003) Acta Cryst. D59, 1016-1019. "Low-resolution molecular replacement using a six-dimensional search".
12. Kollman & Quispe, 2005, J. Struct. Bio., 151, 306-314. “The 17 angstrom structure of the 420kDa lobster clottable protein by single particle reconstruction from cryoelectron micrographs”.
13. Y Xie and Q Hao (1997), Acta Cryst., A53, 643-648. “Evaluation of reflection intensities for the components of multiple Laue diffraction spots by the maximum entropy method
14. Q. Liu, I. A. Kriksunov, R. Graeff, C. Munshi, H. C. Lee, and Q. Hao (2005) Structure, vol. 13, 1331-1339. Crystal Structure of Human CD38 Extracellular Domain. (Cover feature)
15. Love ML, Szebenyi DME,
16. Q. Liu, Q. Huang, X. G. Lei & Q. Hao (2004) Structure, vol 12, 1575-1583. “Crystallographic Snapshots of Aspergillus fumigatus Phytase, Revealing Its Enzymatic Dynamics”.
17. Xiang T, Liu Q, Deacon AM, Koshy
18. C.R. Simmons, Q. Liu, Q.Q. Huang, Q. Hao, T.P. Begley, P.A. Karplus, and M.H. Stipanuk (2006). J. Biol. Chem., 10.1074/jbc.M601555200. Crystal structure of mammalian cysteine dioxygenase: A novel mononuclear iron center for cysteine thiol oxidation.
19. C. R. Simmons, Q. Hao and M. H. Stipanuk (2005) Acta Cryst. F61, 1013-1016. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation.
20. D. J. Schuller, Q. Liu, I. A. Kriksunov, A. M. Campbell, J. Barrett, P. M. Brophy, Q. Hao (2005) Proteins: Structure, Function, and Bioinformatics, vol 61, 1024-1031. Crystal structure of a new class of glutathione transferase from the model human hookworm nematode Heligmosomoides polygyrus.
I.A., Schuller D.J.,
22. X.W. Li, M. Guo, J. Fan, W.Y. Tang, D.Q. Wang, H.H. Ge, H. Rong, M.K. Teng, L.W. Niu, Q. Liu, and Q. Hao (2006) Protein Science. 15:761-773. Crystal structure of 3-hydroxyanthranilic acid 3,4-dioxygenase from Saccharomyces cerevisiae: A special subgroup of the type III extradiol dioxygenase.
23. J. Wang, B. Shen, M. Guo, X. Lou, Y. Duan, X. P. Cheng, M. Teng, L. Niu, Q. Liu, Q. Huang, and Q. Hao (2005). Biochemistry, 44 (30), 10145 –10152. Blocking Effect and Crystal Structure of Natrin Toxin, a Cysteine-Rich Secretory Protein from Naja atra Venom that Targets the BKCa Channel.
24. M. Guo, M. Teng, L. Niu, Q. Liu, Q. Huang, and Q. Hao (2005) J. Biol. Chem., Vol. 280, 12405-12412. “Crystal structure of cysteine-rich secretory protein stecrisp reveals the cysteine-rich domain has a K+channel inhibitor-like fold.”
25. J. Wang, M. Guo, X. Tu, D. Zheng, M. Teng, L. Niu, Q. Liu, Q. Huang and Q. Hao (2004) Acta Cryst. D60, 1108-1111. “Purification, partial characterization, crystallization and preliminary X-ray diffraction of two cysteine-rich secretory proteins from Naja atra and Trimeresurus stejnegeri venoms”.
26. H. Zhang, M. Teng, L. Niu, Y. Wang, Y. Wang, Q. Liu, Q. Huang, Q. Hao, Y. Dong and P. Liu (2004) Acta Cryst. D60, 974-977. “Purification, partial characterization, crystallization and structural determination of AHP-LAAO, a novel L-amino-acid oxidase with cell apoptosis-inducing activity from Agkistrodon halys pallas venom”.
27. U. Englich, A. Kazimirov, Q. Shen, D. H. Bilderback, S. M. Gruner and Q. Hao (2005). J. Synchrotron Rad. 12, 345-348. “Crystallographic data collection using a 0.22% bandwidth multilayer.”
28. A. Kazimirov, D.-M. Smilgies, Q. Shen, X. Xiao, Q. Hao, E. Fontes, D. H. Bilderback, S. M. Gruner, Y. Platonov and V. V. Martynov (2006) J. Synchrotron Rad. 13, 204-210. Multilayer X-ray optics at CHESS.
29. Olczak A. Cianci M., Hao Q., Rizkallah P.J., Raftery J. & Helliwell J.R. (2003) Acta Cryst. A59, 327-334. "Softer-SWAT (Softer-Single Wavelength Anomalous Technique): potential in high-throughput protein crystallography".