Abstract:Fdc1 is a decarboxylase enzyme that requires the novel prenylated FMN cofactor for activity. Here, we use it as an exemplar system to show how native top-down and bottom-up mass spectrometry can measure the structural effect of cofactor binding by a protein. For Fdc1Ubix, the cofactor confers structural stability to the enzyme. IM–MS shows the holo protein to exist in four closely related conformational families, the populations of which differ in the apo form; the two smaller families are more populated in th… Show more
“…The data show that holo-DHODH retains a compact conformation with a CCS of 2,930–3,000 Å 2 , close the value of 2,800 Å 2 expected for the native protein, while apo-DHODH appears to be fully unfolded ( Figure S1 ). We conclude that we are able to preserve compactly folded DHODH with intact co-factor interactions in the gas phase, which may reflect a global stabilizing effect of FMN binding ( Beveridge et al., 2016 ). Figure 1 Non-denaturing MS of DHODH Complexes (A) The nESI-MS spectrum of the full m/z range shows peaks corresponding to apo-DHODH with a broad charge state distribution.…”
SummaryThe interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
“…The data show that holo-DHODH retains a compact conformation with a CCS of 2,930–3,000 Å 2 , close the value of 2,800 Å 2 expected for the native protein, while apo-DHODH appears to be fully unfolded ( Figure S1 ). We conclude that we are able to preserve compactly folded DHODH with intact co-factor interactions in the gas phase, which may reflect a global stabilizing effect of FMN binding ( Beveridge et al., 2016 ). Figure 1 Non-denaturing MS of DHODH Complexes (A) The nESI-MS spectrum of the full m/z range shows peaks corresponding to apo-DHODH with a broad charge state distribution.…”
SummaryThe interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
“…Similar results have been observed for WT IAPP and Aβ40, with mixing resulting in co-polymerization, 32 but seeding being more complex, with Aβ40 fibrils being able to cross-seed hIAPP monomers, but not vice versa . 10 , 85 , 86 The differences are reminiscent of the classic lock and key versus induced fit models of binding. The ESI-IMS-MS studies also reveal a striking relationship between the conformational properties of monomeric IAPP and the length of the lag phase.…”
“…We next used nano-electrospray ionisation (nESI) and ion mobility (IM) mass spectrometry (MS) 52,53 to investigate whether Phe impedes FBP-induced activation of PKM2 by perturbing the oligomeric state of the protein. The mass spectrum of PKM2 apo showed a mixture of monomers, dimers and tetramers at an approximate ratio of 1:7:10 ( Fig.…”
Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands. activity promotes pro-tumorigenic functions, including divergence of glucose carbons into biosynthetic and redox-regulating pathways that support proliferation and defence against oxidative stress 13,14,20,21 . Small-molecule activators, which render PKM2 constitutively active by overcoming endogenous inhibitory cues, attenuate tumour growth, suggesting that regulation of PKM2 activity, rather than increased PKM2 expression per se, is 4 important 13,20,22,23 . Therefore, PKM2 has emerged as a prototypical metabolic enzyme target for allosteric modulators and this has contributed to the renewed impetus to develop allosteric drugs for metabolic enzymes, which are expected to specifically interfere with cancer cell metabolism while sparing normal tissues 11 .The structure of the PKM2 protomer comprises N-terminal, A, B and C domains. Various ligands that regulate PKM2 activity bind to sites distal from the catalytic core, nestled between the A and B domains. The upstream glycolytic intermediate fructose-1,6-bisphosphate (FBP) binds to a pocket in the C-domain 24 and increases the enzymatic activity of PKM2, thereby establishing a feed-forward loop that prepares lower glycolysis for increased levels of incoming glucose carbons. Activation of PKM2 by FBP is associated with a decreased K M for the substrate PEP, while the k cat remains unchanged 25-28 , although some reports also find an elevated k cat 29,30 and the reason for this discrepancy remains unknown. Additionally, several amino acids regulate PKM2 activity by binding to a pocket in the TIM-barrel core 25,31,32 . Lserine (Ser) and L-histidine (His) increase, whereas L-phenylalanine (Phe), L-alanine (Ala), Ltryptophan (Trp), L-valine (Val) and L-proline (Pro) decrease the apparent affinity for PEP.Similar to FBP, the reported effects on k cat vary 25,29,31,33 . Furthermore, succinyl-5aminoimidazole-4-carboxamide-1-ribose 5′-phosphate (SAICAR) 34 ...
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