Welcome to the Komives Lab
Work in the Komives Lab is focused on understanding the parameters that govern protein-protein interactions mediated by non-globular proteins. Four(4) main systems are currently under study.
We were the first lab to do amide H/D exchange to understand what happens to the solvent accessibility of the interface during protein-protein interactions. We developed methods to do H/D exchange by MALDI mass spectrometry, a readily available experimental set-up for non-mass spec experts.
The lab consists of three postdoctoral fellows, six graduate students and one undergrad. Our lab oversees the Biophysics Instrumentation Facility, which contains a BIAcore 3000, a Beckman XL-I Analytical Ultracentrifuge, a Stopped-Flow CD and a MicroCal VP Isothermal Titration Calorimeter. We also oversee the Biomolecular Mass Spectrometry at UCSD which includes a Voyager MALDI-TOF Mass Spectrometer two Q-STAR q-TOF Mass Spectrometers and a MALDI TOF-TOF mass spectrometer. The lab is also a member of the NMR User Group at UCSD.
Thrombin is generated by the coagulation cascade, and it is the enzyme responsible for cleaving fibrinogen to make the fibrin clot. Thrombin has several binding sites: The active site is where proteolytic cleavage occurs; Exosite 1 is where fibrinogen, thrombomodulin, and hirudin (a leach inhibitor of thrombin) bind; Exosite 2 is where heparin binds to accentuate the inhibition by antithrombin III. Our lab has mainly been interested in allosteric communication between Exosite 1 and the active site. We have used amide H/D exchange to map changes in amide exchange that show possible "pathways" of allostery (Figure). We have also used isothermal titration calorimetry to understand the thermodynamics of the allosteric process. Using NMR, we are currently measuring backbone dynamics to gain a deeper view of the allosteric process and to understand the effects of mutations in thrombin.
Thrombomodulin (TM) is a cell-surface receptor that binds thrombin and changes its catalytic activity from pro-coagulant to anticoagulant. Of the six epidermal growth factor-like (EGF) domains in TM, the EGF4-5-6 fragment has full activity. It binds thrombin in Exosite 1 (Figure) but somehow changes the way thrombin interacts with substrates and inhibitors. When TM is bound, thrombin no longer cleaves fibrinogen, instead it cleaves protein C to make activated protein C (aPC) and aPC then cleaves and inactivates Va and VIIIa the two limiting essential cofactors in blood coagulation. We solved the solution structure of the smallest active fragment of TM and using H/D exchange, we showed that TM allosterically regulates thrombin by changing the loops surrounding the active site. Recently, we have been working to make a soluble, fully-active smaller TM molecule and using NMR to understand how the dynamic loops of TM are involved in binding and allosteric affects in thrombin.
The NFκB family of transcription factors responds to inflammatory cytokines with rapid activation of transcription and subsequent signal repression. Much of the system control depends on the unique characteristics of the inhibitor, IκBα, which appears to have complex weakly-folded parts that are critical for function. We have shown that of the six ankyrin repeats (ARs) in IκBα, the fifth and sixth are only completely folded when IκBα is bound to NFκB. These repeats exchange all their amides within one minute and NMR signals are absent for most of AR5 and 6. Single molecule FRET studies have shown that in free IκBα, AR5 and AR6 fluctuate between a compact state and a more extended state but upon binding NFκB, the fluctuations cease. Combined solution biophysical measurements and quantitative protein half-life measurements inside cells have shown that free IκBα is degraded quickly by a ubiquitin-independent proteasome-mediated process Bound IκBα remains extremely stable, and requires phosphorylation and ubiquitinylation for targeted proteasome-mediated degradation. Through a collaboration with G. Ghosh, P. Wolynes, J. Dyson and A. Hoffmann, we are working towards integrating our biophysical understanding of the protein interactions with how signals are processed inside cells.
Our work in mass spectrometry has several facets. We first demonstrated the possibility of measuring amide H/D exchange by MALDI mass spectrometry and we also showed that interaction surfaces on proteins could be mapped by measuring changes in solvent accessibility of amides on the surface of the protein. Lately, we have been working to improve proteomics work-flows through the characterization of additional proteases with alternative substrate specifities for increased proteome coverage and post-translational modification mapping
In the past, our lab worked on the LDL receptor-related proteins. In the future we plan to pursue several new projects involving interactions of repeat proteins.