ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3 which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and non-productively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and non-productive binding by the incorrect substrate bears strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from non-linearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These non-specific interactions likely act to lower the *
Wild type apomyoglobin folds in at
least two steps: the ABGH core
rapidly, followed much later by the heme-binding CDEF core. We hypothesize
that the evolved heme-binding function of the CDEF core frustrates
its folding: it has a smaller contact order and is no more complex
topologically than ABGH, and thus, it should be able to fold faster.
Therefore, filling up the empty heme cavity of apomyoglobin with larger,
hydrophobic side chains should significantly stabilize the protein
and increase its folding rate. Molecular dynamics simulations allowed
us to design four different mutants with bulkier side chains that
increase the native bias of the CDEF region. In vitro thermal denaturation shows that the mutations increase folding stability
and bring the protein closer to two-state behavior, as judged by the
difference of fluorescence- and circular dichroism-detected protein
stability. Millisecond stopped flow measurements of the mutants exhibit
refolding kinetics that are over 4 times faster than the wild type’s.
We propose that myoglobin-like proteins not evolved to bind heme are
equally stable, and find an example. Our results illustrate how evolution
for function can force proteins to adapt frustrated folding mechanisms,
despite having simple topologies.
The binding enthalpies of peptide nucleic acid (PNA) homoduplexes were predicted using a molecular mechanics generalized Born surface area approach. Using the nucleic acid nearest-neighbor model, these were decomposed into sequence parameters which could replicate the enthalpies from thermal melting experiments with a mean error of 8.7%. These results present the first systematic computational investigation into the relationship between sequence and binding energy for PNA homoduplexes and identified a stabilizing helix initiation enthalpy not observed for nucleic acids with phosphoribose backbones.
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