Gerald M. Carlson

Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis.

Mammalian PEPCK2 catalyzes the reversible formation of PEP from OAA and GTP (or ITP) in a divalent cation-dependent reaction (Scheme 1), as was elegantly discussed in the first minireview of this series on PEPCK (1). n n n nSCHEME 1. n nPEPCK-catalyzed interconversion of OAA and PEP. n n n nIn this third minireview, high-resolution crystal structures of mammalian PEPCK are examined to gain insights into the mechanism of PEPCK catalysis, including the reactions reversibility and nucleotide specificity. Regarding reaction reversibility, PEPCK is responsible for regeneration of the high-energy phosphoryl donor PEP from the unstable, activated β-keto acid OAA. When coupled with pyruvate carboxylase, PEPCK reverses the essentially irreversible formation of pyruvate and ATP from PEP and ADP in the glycolytic reaction catalyzed by pyruvate kinase. As illustrated (Fig. 1), PEPCK could achieve this feat by stabilizing the inherently unstable enolate form of pyruvate generated by decarboxylation of OAA (Scheme 1). Stabilization of this intermediate would reduce the energetic cost for phosphoryl transfer by ∼30 kJ mol−1 relative to direct reversal of the pyruvate kinase-catalyzed reaction. An energetic driving force for the pyruvate kinase reaction is the favorable tautomerization of the high-energy enol to its corresponding keto form; in contrast, by stabilizing the enolate, PEPCK could prevent its energetically favorable protonation and tautomerization, allowing phosphoryl transfer to occur. Thus, by stabilizing this intermediate in a high-energy state, the PEPCK reaction would be energetically rendered freely reversible; the crystal structures that will be described indicate that PEPCK does, in fact, stabilize the enolate intermediate. n n n nFIGURE 1. n nDiagram representing the reaction coordinates for the pyruvate kinase-catalyzed (A) and PEPCK-catalyzed (B) reactions. The standard free energy values given are approximate values based upon the average values from a number of literature sources. The ... n n n nThe recent structures of PEPCK from human, rat, and chicken (2–5), the enzymes from Trypanosoma cruzi (6), Anaerobiospirillum succiniciproducens (7), and Corynebacterium glutamicum (8), and earlier work on the isozyme from Escherichia coli (9–13) illustrate that the active-site residues and architecture are well conserved, despite what is rather poor overall sequence homology when comparing members of the ATP- and GTP-dependent families.3 As detailed in this minireview, the cationic environment of the active site, dominated by the juxtaposition of two divalent metal ions and the positioning of lysine and arginine residues, is well suited to allow for the stabilization of the enolate intermediate discussed above and to facilitate phosphoryl transfer. n nAn informative aspect of the PEPCK-catalyzed reaction revealed by the recent structural data on the GTP-dependent isozyme from rat is the illumination of the previously unappreciated role of conformational changes occurring in the active site during the catalytic cycle (5). The most prevalent mobile feature illustrated by the structural work is a 10-residue Ω-loop lid domain whose closure is potentially capable of protecting the enolate intermediate (Fig. 2) (2–5). A similar domain is present in ATP-dependent PEPCK, as represented by the E. coli enzyme, which was the first PEPCK to be structurally characterized (9). The structural data on PEPCK demonstrate that only upon closure of the lid domain are the substrates positioned correctly for catalysis to occur (5). Furthermore, another loop domain, the ubiquitous P-loop or kinase-1a motif in the GTP-dependent PEPCKs, also shows dynamic behavior, adapting various conformations correlated with substrate binding. The potential role of the dynamic P-loop in catalysis is of interest because it contains a reactive cysteine residue that is conserved in all GTP-dependent PEPCKs and whose specific modification has been known for 2 decades to result in the inactivation of the enzyme (14, 15). As described below, recent structural work characterizing the low-energy conformational states that define the reaction coordinate of the enzyme-catalyzed reaction (2–5, 16), considered together with previous biochemical studies, has allowed a relatively detailed picture of the mechanism of catalysis utilized by PEPCK to emerge. Both the role of the positively charged active site and the important conformational changes occurring within that site are discussed in the context of an integrated mechanism for PEPCK-mediated catalysis. n n n nFIGURE 2. n nCrystallographic images defining the chemical reaction path of PEPCK-mediated conversion of OAA to PEP. A schematic drawing to aid in the interpretation of the structural data is presented on the right-hand side of each panel. In the left-hand images, ...

Ca2+-induced structural changes in phosphorylase kinase detected by small-angle X-ray scattering

Phosphorylase kinase (PhK), a 1.3‐MDa (αβγδ)4 hexadecameric complex, is a Ca2+‐dependent regulatory enzyme in the cascade activation of glycogenolysis. PhK comprises two arched (αβγδ)2 octameric lobes that are oriented back‐to‐back with overall D2 symmetry and joined by connecting bridges. From chemical cross‐linking and electron microscopy, it is known that the binding of Ca2+ by PhK perturbs the structure of all its subunits and promotes redistribution of density throughout both its lobes and bridges; however, little is known concerning the interrelationship of these effects. To measure structural changes induced by Ca2+ in the PhK complex in solution, small‐angle X‐ray scattering was performed on nonactivated and Ca2+‐activated PhK. Although the overall dimensions of the complex were not affected by Ca2+, the cation did promote a shift in the distribution of the scattering density within the hydrated volume occupied by the PhK molecule, indicating a Ca2+‐induced conformational change. Computer‐generated models, based on elements of the known structure of PhK from electron microscopy, were constructed to aid in the interpretation of the scattering data. Models containing two ellipsoids and four cylinders to represent, respectively, the lobes and bridges of the PhK complex provided theoretical scattering profiles that accurately fit the experimental data. Structural differences between the models representing the nonactivated and Ca2+‐activated conformers of PhK are consistent with Ca2+‐induced conformational changes in both the lobes and the interlobal bridges.

Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3‐MDa hexadecameric complex, (αβγδ)4. PhK comprises two arched octameric (αβγδ)2 lobes that are oriented back‐to‐back with overall D2 symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three‐dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three‐dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 Å. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90° instead of 68°, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral δ subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca2+ sensitivity to the enzyme, but bind independently of Ca2+. In addition to binding Ca2+, an obligatory activator of PhKs phosphoryltransferase activity, the δ subunits transmit allosteric signals to PhKs remaining α, β, and γ subunits in activating the enzyme. Under mild conditions about 10% of the δ subunits can be exchanged for exogenous CaM. In this study, a CaM double‐mutant derivatized with a fluorescent donor–acceptor pair (CaM‐DA) was exchanged for δ to assess the conformational substates of PhKδ by single molecule fluorescence resonance energy transfer (FRET) ±Ca2+. The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca2+, as observed by alterations of the compact, mid‐length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid‐length and less common compact conformations of PhKδ became less abundant in the presence of Ca2+, with the δ subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaMs Ca2+‐dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKδ subunits were distinct from those of CaM‐DA free in solution, ±Ca2+, as well as from exogenous CaM bound to the PhK complex as δ′. The distinction between δ and δ′ is that the latter binds only in the presence of Ca2+, but stoichiometrically and at a different location in the complex than δ.

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Background: Structural information concerning the phosphorylatable regulatory β subunit of phosphorylase kinase was lacking. Results: Chemical, biochemical, biophysical, and computational approaches revealed secondary, tertiary, and quaternary structures for this subunit. Conclusion: The β subunit is helical and forms the β4-bridged core in the (αβγδ)4 kinase complex. Significance: These findings reveal the architecture of the complex, which provides an explanation for the conformational changes in its bridged core associated with activating β-phosphorylation. Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)4 complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)2 lobes joined with D2 symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)4 complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)4 kinase core, because a β4 subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle.

Skeletal muscle phosphorylase kinase (PhK) is a Ca(2+)-dependent enzyme complex, (alpha beta gamma delta)(4), with the delta subunit being tightly bound endogenous calmodulin (CaM). The Ca(2+)-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the excitation-contraction-energy production triad. Although the Ca(2+)-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic gamma and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the gamma subunit sharing high sequence similarity with the Ca(2+)-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca(2+) stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca(2+) of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca(2+)-binding proteins troponin C and CaM.

Physicochemical changes in phosphorylase kinase associated with its activation

Phosphorylase kinase (PhK) regulates glycogenolysis through its Ca2+‐dependent phosphorylation and activation of glycogen phosphorylase. The activity of PhK increases dramatically as the pH is raised from 6.8 to 8.2 (denoted as ↑pH), but Ca2+ dependence is retained. Little is known about the structural changes associated with PhKs activation by ↑pH and Ca2+, but activation by both mechanisms is mediated through regulatory subunits of the (αβγδ)4 PhK complex. In this study, changes in the structure of PhK induced by ↑pH and Ca2+ were investigated using second derivative UV absorption, synchronous fluorescence, circular dichroism spectroscopy, and zeta potential analyses. The joint effects of Ca2+ and ↑pH on the physicochemical properties of PhK were found to be interdependent, with their effects showing a strong inflection point at pH ∼7.6. Comparing the properties of the conformers of PhK present under the condition where it would be least active (pH 6.8 − Ca2+) versus that where it would be most active (pH 8.2 + Ca2+), the joint activation by ↑pH and Ca2+ is characterized by a relatively large increase in the content of sheet structure, a decrease in interactions between helix and sheet structures, and a dramatically less negative electrostatic surface charge. A model is presented that accounts for the interdependent activating effects of ↑pH and Ca2+ in terms of the overall physicochemical properties of the four PhK conformers described herein, and published data corroborating the transitions between these conformers are tabulated.

Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhKs activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.

What Mutagenesis Can and Cannot Reveal About Allostery

Allosteric regulation of protein function is recognized to be widespread throughout biology; however, knowledge of allosteric mechanisms, the molecular changes within a protein that couple one binding site to another, is limited. Although mutagenesis is often used to probe allosteric mechanisms, we consider herein what the outcome of a mutagenesis study truly reveals about an allosteric mechanism. Arguably, the best way to evaluate the effects of a mutation on allostery is to monitor the allosteric coupling constant (Qax), a ratio of the substrate binding constants in the absence versus presence of an allosteric effector. A range of substitutions at a given residue position in a protein can reveal when a particular substitution causes gain-of-function, which addresses a key challenge in interpreting mutation-dependent changes in the magnitude of Qax. Thus, whole-protein mutagenesis studies offer an acceptable means of identifying residues that contribute to an allosteric mechanism. With this focus on monitoring Qax, and keeping in mind the equilibrium nature of allostery, we consider alternative possibilities for what an allosteric mechanism might be. We conclude that different possible mechanisms (rotation-of-solid-domains, movement of secondary structure, side-chain repacking, changes in dynamics, etc.) will result in different findings in whole-protein mutagenesis studies.

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: (1) hydrogen/deuterium exchange (HDX), (2) limited proteolysis, and (3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography.