Structure dictates function and with state-of-the-art technologies including high-field NMR and cyro-crystallography we are able to discern macromolecular structures as well as their dynamic fluctuations. Here in the Department of Pharmacology, these techniques are coupled with computational techniques including molecular dynamics and virtual screening to guide drug discovery and to understand the complex interplay of networked signaling cascades.
|Ceftriaxone resistance in N. gonorrhoeae||Signal transduction cascades|
The penA gene in Neisseria gonorrhoeae encodes penicillin-binding protein 2 (PBP 2), an essential peptidoglycan transpeptidase that is the lethal target of the β-lactam antibiotics, penicillin and ceftriaxone. Because N. gonorrhoeae is a naturally competent organism, it can take up DNA from related Neisseria species and recombine regions of that DNA into its genome such that certain genes become “mosaic”. Recently, strains have emerged from the Pacific Rim and spread throughout the world with high-level resistance to ceftriaxone, the last remaining effective antibiotic for gonococcal infections. While multiple mutations are involved in high-level resistance, the most important determinant in these strains is a mosaic penA gene. Mosaic penA alleles contain multiple blocks DNA from different species, resulting in the production of a PBP 2 variant with up to 70 amino acid changes relative to wild-type and a markedly lower rate of acylation by ceftriaxone. Because β-lactam antibiotics are structural analogs of the peptidoglycan substrate that participates in transpeptidation, the changes must alter reactivity to the antibiotic without compromising the capacity of the enzyme to catalyze transpeptidation. We have solved the structure of PBP 2 (see left panel of Figure; red spheres represent the location of the alterations in a mosaic PBP 2 compared to wild-type), and are currently utilizing structural biology, mutagenesis, and biochemistry to identify the amino acid changes that are responsible for decreasing its reactivity to ceftriaxone, and also to determine the mechanism by which this occurs. We recently showed that three mutations in the mosaic penA gene from H041, a high-level ceftriaxone-resistant strain, are responsible for the increase in resistance over intermediate-level resistant strains (right panel of Figure).
Shown below is an expanded view of the active site of an alpha subunit of a heterotrimeric G protein in complex with phospholipase C-β3. This high resolution structure (Waldo, et. al., Science, 2010) explains the reciprocal regulation of these two classes of enzymes operating downstream of a myriad of G protein-coupled receptors and designed for rapid responses with high dynamic range and exquisite noise suppression. Our lab uses diverse biophysical methods to understand and manipulate signaling cascades controlled by G proteins and small GTPases. More recent work includes the uses of peptidomimetics to inhibit signaling by Gαq in uveal melanoma; the creation and use of biosensors to track the spatiotemporal activation of Gα subunits and RhoGEFs; the development of high-throughput screens to identify small molecule inhibitors of Gα subunits and RhoGEFs; and proteomics methods to map the network dynamics of activation of RhoGEFs in breast cancer.
Tomberg J, Unemo M, Ohnishi M, Davies C, Nicholas RA. Identiﬁcation of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041. Antimicrob Agents Chemother 57, 3029-3036 (2013).
Unemo, M., Golparian, D., Nicholas, R., Ohnishi, M., Gallay, A., and Sednaoui, P. High-level cefixime- and ceftriaxone-resistant N. gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 56, 1273-1280 (2011).
Tomberg, J., Unemo, M., Davies, C., and Nicholas, R. A. Molecular and structural analysis of mosaic variants of penicillin-binding protein 2 conferring decreased susceptibility to expanded-spectrum cephalosporins in Neisseria gonorrhoeae: role of epistatic mutations. Biochemistry49, 8062-8070 (2010).
Waldo GL, Ricks TK, Hicks SN, Cheever ML, Kawano T, Tsuboi K, Wang X, Montell C, Kozasa T, Sondek J, Harden TK. Kinetic scaffolding mediated by a phospholipase C-b and Gq signaling complex. Science 330, 974-980, (2010).
Hicks SN, Jezyk MR, Gershburg S, Seifert JP, Harden TK, Sondek J. General and versatile autoinhibition of PLC isozymes. Mol Cell 31, 383-394, (2008).
Cheever ML, Snyder JT, Gershburg S, Siderovski DP, Harden TK, Sondek J. Crystal structure of the multifunctional Gbeta5-RGS9 complex. Nat Struct Mol Biol 15, 155-162, (2008).
Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nat Struct Mol Biol 14, 814-823, (2007).