Mauricio Baez

The Crystal Complex of Phosphofructokinase-2 of Escherichia coli with Fructose-6-phosphate: KINETIC AND STRUCTURAL ANALYSIS OF THE ALLOSTERIC ATP INHIBITION.

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K0.5 for fructose-6-P and a decrease in the apparent kcat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (nH of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.

Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli

In a previous work, chemical modification of Cys‐238 of Escherichia coli Pfk‐2 raised concerns on the importance of the dimeric state of Pfk‐2 for enzyme activity, whereas modification of Cys‐295 impaired the enzymatic activity and the MgATP‐induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk‐2 is critical for the stability and the activity of the enzyme. The replacement of Cys‐238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk‐2. However, the mutation of Cys‐295 by either Ala or Phe provokes a decrease in the k cat value and an increment in the K m values for both substrates. We suggest that the Cys‐295 residue participates in intersubunit interactions in the tetramer since the Cys‐295‐Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose‐6‐P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.

Effect of mutation at the interface of Trp-repressor dimeric protein: a steered molecular dynamics simulation

The strength of key interfacial contacts that stabilize protein–protein interactions have been studied by computer simulation. Experimentally, changes in the interface are evaluated by generating specific mutations at one or more points of the protein structure. Here, such an evaluation is performed by means of steered molecular dynamics and use of a dimeric model of tryptophan repressor and in-silico mutants as a test case. Analysis of four particular cases shows that, in principle, it is possible to distinguish between wild-type and mutant forms by examination of the total energy and force–extension profiles. In particular, detailed atomic level structural analysis indicates that specific mutations at the interface of the dimeric model (positions 19 and 39) alter interactions that appear in the wild-type form of tryptophan repressor, reducing the energy and force required to separate both subunits.

Reversible unfolding of dimeric phosphofructokinase-2 from Escherichia coli reveals a dominant role of inter-subunit contacts for stability.

Escherichia coli phosphofructokinase‐2 (Pfk‐2) is a homodimer whose subunits consist of a large domain and an additional β‐sheet that provides the interfacial contacts between the subunits, creating a β‐barrel flattened‐like structure with the adjacent subunits β‐sheet. To determine how the structural organization of Pfk‐2 determines its stability, the reversible unfolding of the enzyme was characterized under equilibrium conditions by enzymatic activity, circular dichroism, fluorescence and hydrodynamic measurements. Pfk‐2 undergoes a cooperative unfolding/dissociation process with the accumulation of an expanded and unstructured monomeric intermediate with a marginal stability and a large solvent accessibility with respect to the native dimer.

Uncoupling the MgATP-induced inhibition and aggregation of Escherichia coli phosphofructokinase-2 by C-terminal mutations

Binding of MgATP to an allosteric site of Escherichia coli phosphofructokinase‐2 (Pfk‐2) provoked inhibition and a dimer–tetramer (D–T) conversion of the enzyme. Successive deletions of up to 10 residues and point mutations at the C‐terminal end led to mutants with elevated K Mapp values for MgATP which failed to show the D–T conversion, but were still inhibited by the nucleotide. Y306 was required for the quaternary packing involved in the D–T conversion and the next residue, L307, was crucial for the ternary packing necessary for the catalytic MgATP‐binding site. These results show that the D–T conversion could be uncoupled from the conformational changes that lead to the MgATP‐induced allosteric inhibition.

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner.

Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions ☆

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units per oocyte in the absence of glucose-6-P. V(max) in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 microM and, depending on substrate concentrations, 50-100 microM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.

Knots can impair protein degradation by ATP-dependent proteases

Significance AAA+ proteases are ring-shaped machines with a central narrow pore used to translocate protein substrates into a proteolytic chamber. Knotted proteins, however, may block the narrow pore by formation of an incompressible tight knot. Here, we determined how the archetypal AAA+ ClpXP protease degrades a protein substrate containing a trefoil knot. A trefoil knot is degraded quickly by ClpXP but not when it is fused to GFP placed downstream of the knot. Specifically, ClpXP releases partially degraded products when a tight knot is buttressed against GFP. Furthermore, degradation of substrates harboring linkers of different lengths between the knot and GFP allowed us to explore the dynamics of knot tightening and sliding and to postulate a new biological role for knotted proteins. ATP-dependent proteases translocate proteins through a narrow pore for their controlled destruction. However, how a protein substrate containing a knotted topology affects this process remains unknown. Here, we characterized the effects of the trefoil-knotted protein MJ0366 from Methanocaldococcus jannaschii on the operation of the ClpXP protease from Escherichia coli. ClpXP completely degrades MJ0366 when pulling from the C-terminal ssrA-tag. However, when a GFP moiety is appended to the N terminus of MJ0366, ClpXP releases intact GFP with a 47-residue tail. The extended length of this tail suggests that ClpXP tightens the trefoil knot against GFP, which prevents GFP unfolding. Interestingly, if the linker between the knot core of MJ0366 and GFP is longer than 36 residues, ClpXP tightens and translocates the knot before it reaches GFP, enabling the complete unfolding and degradation of the substrate. These observations suggest that a knot-induced stall during degradation of multidomain proteins by AAA proteases may constitute a novel mechanism to produce partially degraded products with potentially new functions.

The Folding Unit of Phosphofructokinase-2 as Defined by the Biophysical Properties of a Monomeric Mutant

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the β-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the β-meander module is not affected by monomerizing mutations.

Folding kinetic pathway of phosphofructokinase-2 from Escherichia coli: A homodimeric enzyme with a complex domain organization

Pfk‐2 binds to Pfk‐2 by circular dichroism (View interaction)