CONCLUSIONS AND FUTURE DIRECTIONS
The nature of the Pdx-CYP101 interaction and its role in Pdx effector activity has mystified researchers in the P450 area for quite some time. The work performed in this thesis is an important first step in answering some fundamental questions on how these two proteins interact in a redox-dependent manner. Conclusive evidence from this study demonstrates that Pdxo and Pdxr bind to same site on the proximal face of CYP101o and CYP101r respectively, with 1:1 stoichiometry. Since the Pdx binding site and the complex structures are the same between the two redox complexes, redox-dependent effector activity is more likely to be the result of redox-modulated conformational dynamics. Given these findings, it is unlikely that different electron transfer pathways exist between the redox complexes as has been previously speculated.
An important question still remains in how Pdx binding propagates redox- dependent changes to the substrate access channel. Although the recent co- crystal structures argue in favor of an open conformation for both redox states at the F-G loop and I-Helix which activates and orchestrates the catalytic machinery, the role of dynamics in modulating some of these changes cannot be understated. While not definitive, our data supports the conformational dynamics model of Pdx effector activity that postulates the Pdx effector mechanism works through a redox-modulated selection of a closed subset of conformations. This closed conformation is hypothesized to enhance catalysis by preventing substrate release prior to electron transport and product turnover. The farther distances measured at the B’-helix in the reduced complex structure are representative of the closed conformational subset.
The in-solution PRE measurements have an attribute of reporting on the dynamic ensemble of conformations, effectively providing information on the average conformational distribution over the entire length of the experiment.
structures, a severe limitation is placed on the structure calculation by foregoing the dynamic ensemble nature of the complex structure. Small conformational changes upon binding are also quite difficult to model correctly in silico with the small number of PRE restraints that were used in docking the two proteins. Deciding whether differences between the measured and calculated distances are a result of experimental error or conformational changes becomes an ambiguous exercise in this case. The minor changes witnessed in the 15N HSQC- TROSY spectra of CYP101 before and after binding of Pdx, indicate no major secondary structure changes take place, and the recent crystal structures corroborate this observation. Therefore, trying to capture the subtle conformational differences and in particular, the dynamic differences, that, in all likelihood, define the Pdx effector mechanism may require a more sensitive method than PRE.
There are some additional ways in which this complex structure model can be improved. One possibility is to include orientational information in the structure calculation, to refine the relative orientation of the two proteins, which currently is only a rough approximation. Orientational restraints in the form of residual dipolar couplings (RDC) can be experimentally measured and used in concert with a more sophisticated structure calculation program such as the HADDOCK docking simulation software (69), which can incorporate both PRE and RDC data. This has already been shown as a feasible approach in the structural modeling of the oxidized complex and can be extended to the reduced complex by modification of parameter and topology files for the metal cofactors in the reduced state of each protein. Efforts are currently ongoing in our group to collect these orientational restraints and utilize them in such a structure calculation protocol. Incorporation of RDCs is bound to increase the accuracy of the local structure and provide a means to elucidate the minute conformational differences that exist between the oxidized and reduced complexes in solution. More importantly, measurement of orientational constraints in multiple alignment media would enable us to measure even the dynamic changes that accompany these
conformational changes, since RDCs are sensitive to atomic motions on all timescales.
Uncovering the effector mechanism will likely require a better characterization of the dynamic differences between the two redox complexes. Such dynamic characterization is particularly important for perturbations upon complexation of highly dynamic regions such as the B’-Helix and the F-G Loop, that are observed in both our NMR study and the co-crystal structures (29). Conformational exchange in these secondary structures is expected to occur on the micro-millisecond timescales (70). Apart from the RDC measurements, chemical exchange experiments based on CPMG sequences can also provide helpful dynamic information on motions occurring in the micro- millisecond time regime (45). These experiments should also allow collecting dynamic information on regions inaccessible to our PRE experiments. If we can determine the exact nature of the conformation in each redox state for the complex in solution i.e. open or closed, then it may be possible to comprehensively describe the redox- modulated conformational changes propagated throughout CYP101 due to effector binding with the help of molecular dynamics (MD) simulations. These simulations have the potential of capturing differences in the conformational sampling of various states within the subtle secondary structure motions in the F- G Loop and B’-Helix that are responsible for substrate gating. Extracting redox- modulated exchange rates in these regions could provide compelling evidence in support of the conformational dynamics model of Pdx effector activity.
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Table 1. PRE Measurements for Pdxr and back calculated distances from
Pdxr crystal structure (PDB I.D. 1XLQ)
Residue no. Measured (Å) Calculated (Å)
3 32.8 34.8 4 32.9 33.7 6 30.3 31.4 7 29.7 31.8 8 30.3 31.9 9 31.5 30.4 18 32.5 31 21 32.3 30.7 22 28.4 27.9 31 32.5 33.4 33 29.4 30.1 35 28.2 28.3 51 27.5 26.1 52 25.2 23.7 53 24.9 24.8 54 29.4 28.1 56 31 29.4 58 30.7 30.6 58 29.7 28.2 59 29.8 29.4 60 30.6 29.1 62 28.3 27.5 63 24.6 22.7 78 23.5 19.1 79 22.6 21 81 24.1 22.4 83 24.4 20.5 90 31.2 29.3 89 28.2 27.3 103 25.5 25.6 105 23.7 21.8 106 22.6 20.4 a
Distances are measured from the intensity ratios calculated for the HN backbone amide
resonances of 15N labeled Pdxr conjugated with EDTA spin label and coordinated to Gd3+. Error
VITA
Nicholas John Lopes was the second of three sons born to Anthony and Cathy Lopes on August 9, 1985 in Miami, FL. At six months of age he moved with his family to Germantown, TN, a quaint suburb of Memphis, TN. There he attended public school at Dogwood Elementary and Houston Middle, before receiving a college-preparatory education at Christian Brothers High School, an all-boy, catholic school of the Lasallian tradition. Immediately following high school graduation in 2004, he enrolled at the University of Tennessee, Knoxville (UTK). After vacillating between majors for a year he settled on Ecology and Evolutionary Biology. It was his decision to volunteer at a microbiology laboratory in the Center for Environmental Biotechnology (CEB) at UTK during his junior year that set him on the path of academic research. While continuing his work at the CEB, Nicholas received his Bachelor’s degree in 2008. After spending one additional year as a research technician with the CEB, he accepted an offer to enter the Biochemistry, Molecular and Cellular Biology Department graduate program at UTK. Upon receiving his Master’s degree he hopes to continue pursuing his dream of leading an interesting life.