With an eye toward developing new methods, our work focuses on two areas: understanding host-pathogen interactions and discovering the nature and the functions of motions in proteins. Our studies of infectious diseases aim to define mechanisms of signaling and host interactions in TB and HIV/AIDS, two of the major causes of worldwide mortality. Our explorations of protein motions are based on the discovery that X-ray crystallography can reveal a huge ensemble of alternate structures that map out functional dynamics. We use a variety of experimental and computational approaches ranging from synchrotron crystallography and biochemistry to molecular biology and genetics.
To cause disease, pathogens sense their environment and manipulate host cell processes. Our work is aimed at defining the fundamental mechanisms of sensing and signaling in M. tuberculosis. In addition, we are studying the interactions of HIV proteins with human partners discovered in collaboration with the Zhou (UC Berkeley) and Krogan (UCSF) groups. To explore the fundamental aspects of protein motions, we are developing new methods to model structural ensembles from crystallographic data.
Regulatory mechanisms in infectious disease. A major goal is to understand signaling pathways in Mycobacterium tuberculosis (Mtb). This pathogenic bacterium infects one third of the world's population and causes over two million deaths annually, more than any other infectious disease. Through reversible protein phosphorylation, protein kinases and phosphates provide critical machinery for environmental sensing and physiological signaling. In addition to the traditional two-component signaling systems, Mtb expresses 11 "eukaryotic-like" Ser/Thr protein kinases (STPKs) and four protein phosphates that are candidate mediators of developmental changes and host interactions. The biological functions of these systems are largely unknown.
In pioneering structural and mechanistic studies of these Ser/Thr/Tyr signaling systems, we found that universal mechanisms of regulation and substrate recognition govern the functions of prokaryotic and eukaryotic STPKs. These studies also revealed novel mechanisms of regulation, including dimerization of STPK domains and a dynamic lid that protects the PtpB Tyr phosphatase from oxidative inactivation in host cells. Our immediate challenges are to discover the networks of kinase crosstalk in Mtb and to define how the master kinase, PknB, regulates cell-wall homeostasis and cell growth. In these studies, we have used a novel regulated protein knockdown system developed in collaboration with the Rubin (Harvard School of Public Health) and Sassetti (U Mass Medical School) groups to selectively degrade proteins in vivo. Recent work revealed the first structure of a bacterial pseudokinase and showed how phosphorylation of this module regulates the transport of peptidoglycan precursors and the integrity of the cell wall. Our future work focuses on defining the signaling pathways of the Mtb STPKs in vivo and discovering the mechanisms of physiological regulation by post-translational modifications.
The pandemics of TB and AIDS are closely associated, and despite nearly three decades of intensive work, major gaps in knowledge remain about how HIV hijacks host cell functions. Following an unbiased proteomics approach with the Krogan and Zhou labs, we discovered new host partners of the viral proteins. These new interacting factors include a transcriptional elongation complex recruited to the HIV promoter by HIV Tat. To test the hypothesis that Tat allosterically regulates transcriptional elongation, we are mapping interactions in this large complex and assembling subcomplexes for biochemical and structural studies.
Protein polymorphism. Protein function depends on a balance between structural rigidity and flexibility. We developed two new methods, including the Ringer program (http://ucxray.berkeley.edu/ringer.htm), for defining conformational polymorphism in protein crystal structures. Our analysis of >400 available structures reveals that native proteins adopt a large number of specific, alternate conformations that previously escaped detection. Our results indicate that crystalline proteins are much more polymorphic than current crystallographic models. In proline isomerase, we found using unorthodox room-temperature X-ray diffraction measurements that coupled shifts define a “dynamic network” that determines the catalytic rate. Coupled structural changes also may mediate intramolecular signaling pathways, while uncoupled conformations contribute to residual entropy that favors protein folding or ligand binding. Modeling ensembles and exploring their functional significance for enzyme mechanisms, protein recognition and drug binding represent fruitful directions for ongoing work.
Lang, P.T., Ng, H.L., Fraser, J.S., Corn, J.E., Echols, N. Sales, M., Holton, J.M., Alber, T. (2010). Automated electron-density sampling reveals widespread conformational polymorphism in proteins. Protein Science 19, 1420-31.
Prach, L., Kirby, J., Keasling, J.D., Alber, T. (2010). Diterpene production in M. tuberculosis, FEBS J, July 29, 2010.
Flynn, E.M., Hanson, J.A., Alber, T., Yang, H. (2010). Dynamic active-site protection by the M. tuberculosis protein tyrosine phosphatase PtpB lid domain. J Amer Chem Soc 132, 4772-80.
He, N., Liu, M., Hsu, J., Xue, Y., Chou, S., Burlingame, A., Krogan, N.J., Alber, T., Zhou, Q. (2010). HIV-1 Tat and host cellular AFF4 recruit two distinct transcription elongation factors into a bifunctional elongation complex for coordinated activation of HIV-1 transcription. Mol Cell, 38, 428-438.
Alber, T. (2009). Signaling mechanisms of the Mycobacterium tuberculosis receptor Ser/Thr protein kinases. Current Opinion Struct Biol 19, 650-57.
Fraser, J.S., Clarkson, M.W., Degnan, S.C., Erion, R., Kern, D., Alber, T. (2009). Hidden alternative structures of proline isomerase essential for catalysis, Nature, 462, 669-672.
Greenstein, A.E., Hammel, M., Cavazos, A. Alber, T. (2009). Interdomain communication in the Mycobacterium tuberculosis environmental phosphatase Rv1364c. J Biol Chem 284, 29828-35.
Rawls, K.A., Lang, P.T, Takeuchi, J., Imamura, S, , et al. (2009). Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA. Bioorg Med Chem Lett 19, 6851-54.
Last Updated 2010-08-28