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Crossing Over, Genetic↗
Biomedical subjects
J N Pelletier
Publications and source records attributed to J N Pelletier.
A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble.
Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.
Amino Acid Sequence↗
Detection of protein-protein interactions by protein fragment complementation strategies.
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Animals↗
An in vivo library-versus-library selection of optimized protein-protein interactions.
We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.
Animals↗
Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments.
Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.
Animals↗
Crystallization of the bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase domain of the human trifunctional enzyme.
Methylenetetrahydrofolate([H4] folate) dehydrogenase (D) and methenyl[H4] folate cyclohydrolase (C) coexist as a bifunctional enzyme (DC) or as the amino-terminal domain of a trifunctional enzyme (DCS) where the third activity is 10-formyl[H4]folate synthetase (S). Two crystal forms of the DC domain of the human cytosolic DCS enzyme have been grown from polyethyleneglycol solution. The monoclinic P2(1) crystals diffract to 2.8 A with a = 72.5 A, b = 68.5 A, c = 125.2 A, and beta = 91.8 degrees but were found to be twinned. The orthorhombic P2(1)2(1)2(1) crystals diffract to 2.5 A with a = 67.7 A, b = 135.9 A, c = 61.6 A, and contain two molecules per asymmetric unit.
Aminohydrolases↗
Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
The bifunctional dehydrogenase/cyclohydrolase domain of the human NADP-dependent trifunctional methyleneH4folate dehydrogenase/methenylH4folate cyclohydrolase/formylH4folate synthetase (H4folate = tetrahydrofolate) catalyzes two sequential reactions involved in the interconversion of H4folate derivatives. We have established by equilibrium dialysis that a single H4folate-binding site exists per monomer of the dimeric domain and that the presence of nucleotides has two unexpected effects on H4folate substrate binding. Nucleotides containing a 5'-phosphate cause positive cooperativity in the binding of methyleneH4folate but not of 10-formylH4folate, and NADP increases the affinity for 10-formylH4folate by a factor of 25. The results indicate that dinucleotide preferentially binds before 10-formylH4folate in the reverse cyclohydrolase reaction, and this mechanism increases the efficiency of conversion of 10-formylH4folate to methyleneH4folate. We report new kinetic data that are also consistent with a steady-state random mechanism for this enzyme. To assess whether the enzyme functions at equilibrium in vivo, we determined the overall chemical equilibrium constant of Keq = 16 for ([10- formylH4folate][NADPH])/([methyleneH4folate][NADP]). Using this value and reported ratios of free dinucleotides and folate derivatives in vivo, we estimate that the cytosolic dehydrogenase/cyclohydrolase reactions exist near the equilibrium position. However, the NAD-dependent dehydrogenase/cyclohydrolase reactions in mitochondria are far from equilibrium and are poised toward 10-formylH4folate synthesis. The results of the binding and kinetic studies indicate that the bifunctional nature of the methyleneH4folate dehydrogenase/methenylH4folate cyclohdrolase domain is designed to optimize the overall reverse reactions in vivo.
Aminohydrolases↗
Binding of the 2',5'-ADP subsite stimulates cyclohydrolase activity of human NADP(+)-dependent methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
The bifunctional dehydrogenase/cyclohydrolase domain of the human trifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase catalyzes two sequential reactions with significant channeling of the intermediate, methenyltetrahydrofolate. Equilibrium dialysis established that a single, high-affinity NADP+ binding site exists per monomer of the dimeric enzyme. Kinetic characterization of NADP+ binding to the dehydrogenase using analogs as inhibitors demonstrated that affinity for this substrate is due almost exclusively to binding at the 2',5'-ADP subsite. The same structural specificities for binding are exhibited by these analogs in their effects on the cyclohydrolase. Both NADP+ and its 3-aminopyridine analog AADP partially inhibit the activity of the cyclohydrolase when assayed with added methenyltetrahydrofolate as substrate. However, under the same conditions, the cyclohydrolase is actually activated by 2',5'-ADP; activation requires the presence of the 5'-phosphate since 2'-AMP binds but does not activate. Nicotinamide ribose monophosphate (NMN) has no detectable effect either alone or in combination with 2',5'-ADP. The results are consistent with the existence of a shared dehydrogenase/cyclohydrolase active site proximal to the 2',5'-ADP subsite. NADP+ reduces the rate of the fully activated cyclohydrolase by 2-fold. Inhibition appears to be due to the loosely bound nicotinamide ring interacting with the common folate subsite, resulting in only partial inhibition by NADP+. The interaction of 2',5'-ADP with the cyclohydrolase suggests a potential role for this portion of the molecule in promoting the efficiency of the channeling of endogenously generated methenyltetrahydrofolate.
Adenine Nucleotides↗