Article pubs.acs.org/biochemistry
Asymmetric Anchoring Is Required for Efficient Ω‑Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase Danica S. Cui,†,§ Aron Broom,‡ Matthew J. Mcleod,† Elizabeth M. Meiering,‡ and Todd Holyoak*,† †
Department of Biology and ‡Department of Chemistry, University of Waterloo, Waterloo, ON, Canada N2L 3G1 S Supporting Information *
ABSTRACT: Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the Nterminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ωloop by 3.4 kcal mol−1. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in kcat for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state. he Ω-loop is a prevalent, nonregular element of secondary structure seen in many enzymes that has the capacity to play different functional roles such as protein folding, substrate binding, and catalytic function.1−4 It is typically found on the surface of the enzyme with a backbone consisting of 10−20 amino acids forming an Ω-shaped three-dimensional loop structure and being defined by having a short distance between the entry and exit points from the body of the structure (3.7−10 Å).5,6 Following the definition of a nonregular structural element, the mobile Ω-loop structure is not defined by distinguished dihedral angles and contains little or no internal hydrogen bonding, allowing it to be flexible in its nature.5 Perhaps the most comprehensively studied Ω-loop is that of loop 6 from triosephosphate isomerase (TIM).4,7−12 Composed of 11 residues, this loop structure has been found to undergo a transition between open and closed conformations as a rigid domain pivoting on three-residue hinges located at the N- and Cterminal ends of the mobile loop. In addition, the evolutionarily conserved hinge sequences have been demonstrated to reflect a key design strategy providing a balance between flexibility and rigidity that allow the rigid lid domain to efficiently undergo the transition between the two catalytically required conformational states in a focused trajectory, minimizing the sampling of nonproductive conformational states.10 In the enzyme under study here, phosphoenolpyruvate carboxykinase (PEPCK), the Ω-loop domain is found directly over the active site, and like loop 6 in TIM, the loop has been demonstrated to be essential to catalytic function.13−16 However, in contrast to loop 6 in TIM, the Ω-loop lid of PEPCK is essential
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© XXXX American Chemical Society
to catalysis despite containing no residues that interact directly with the substrates or products of the reaction. As suggested by the crystallographic data, two extreme conformations of the loop exist. The lid appears to occupy an open conformation when no substrates are present and undergoes a transition toward a closed, catalytically active state after the formation of the Michaelis complex.16−18 Prior studies have resulted in the conclusion that the lid domain plays an essential role in PEPCK catalysis by (i) stabilizing the global closure of the N- and C-terminal lobes of the enzyme, a process that reduces the active site volume and correctly positions the substrates for catalysis, and (ii) protecting the reactive intermediate enolate from spontaneous protonation during catalysis.19 In the GTP-dependent family of PEPCKs from the phylum chordata, the Ω-loop sequence is highly conserved (Figure 1) . In those members of this grouping that have been structurally characterized (human, rat, and chicken), the closed Ω-loop spans E463−I475 (numbering is that for the rat cytosolic enzyme, cPEPCK) where it is preceded and followed by a short twostranded β-sheet consisting of residues 461−463 and 475−477, respectively (Figure 2). In the closed lid conformation, residues R461 and H477 are observed to form hydrogen bonds and ionic interactions with the penultimate helix in the C-terminal lobe of the enzyme through interactions with E588 and E591, Received: February 27, 2017 Revised: March 24, 2017 Published: March 27, 2017 A
DOI: 10.1021/acs.biochem.7b00178 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Figure 1. Sequence logo showing the conservation in the Ω-loop lid region of GTP-dependent PEPCK from the phylum chordata (PFAM:00821). The Ω-loop spans residues E463−I475 and is preceded and followed by two short β-strands (residues R461−E463 and I475−H477). Sequences were downloaded from the PFAM database, manually curated, and aligned with Clustal omega, and a sequence logo was generated from the alignment using Weblogo (http://weblogo.berkeley.edu/).21 The sequence numbering corresponds to that of rat cPEPCK.
conformational transition pathway in which the entire loop is involved in a conformational exchange between a disordered open and a more ordered closed state. This could perhaps be categorized as a folding event rather than a loop functioning with a hinged motion.
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MATERIALS AND METHODS Materials. Dithiothreitol and isopropyl β-D-1-thiogalactopyranoside were purchased from Gold Bio-Tech. The nucleotides (GTP and GDP), PEP, and OAA were from Sigma. NADH was from Chem-Impex, and β,γ-methyleneguanosine 5′-triphosphate (GMPPCP) was from Jena Biosciences. βSP was synthesized and purified as previously described.22 HiQ, P6DG, and Chelex resins were from Bio-Rad. All other chemicals were of the highest grade available from Fisher Scientific. Enzymes. Malate dehydrogenase (22976 units/mL of 50% glycerol solution) and lactate dehydrogenase (6000 units/mL, AmSO4 suspension) were from Calzyme Laboratories. Pyruvate kinase (10 mg/mL, AmSO4 suspension) was from Roche. Mutagenesis. The gene for rat cytosolic PEPCK previously cloned into the pSUMO vector14 was used as the template to generate the H477R mutation. The Stratagene Quik Change protocol was used to perform site-directed mutagenesis using forward primer 5′-GCAAGGTCATCATGCGCGACCCCTTCGCTAT-3′ and reverse primer 5′-ATAGCGAAGGGGTCGCGCATGATGACCTTGC-3′. The mutated DNA was subsequently isolated using a GeneJet plasmid miniprep kit (Thermo Scientific). The sequencing was conducted at The Center for Applied Genomics of The Hospital for Sick Children (Toronto, ON). The pSUMO-cPEPCK(H477R) plasmid was subsequently transformed into BL-21(DE3) electro-competent cells. Protein Expression and Purification. H477R PEPCK protein expression and purification were performed as previously described with the following alterations.14 The cells were lysed by emulsification instead of using a French pressure cell. The supernatant containing PEPCK was incubated with Qiagen NiNTA resin, and the column was washed with 25 mM HEPES (pH 7.5), 300 mM NaCl, 10 mM imidazole, 10% glycerol, and 2 mM TCEP until an A280 of
