Sunghyuk Lim

A Sialyltransferase Mutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities for Directly Sialylating Lewisx

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

Molecular Structure and Target Recognition of Neuronal Calcium Sensor Proteins

BACKGROUND Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulate synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression. SCOPE OF REVIEW Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase, NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. MAJOR CONCLUSIONS We propose the idea that N-terminal myristoylation is critical for shaping each NCS family member into a unique structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues, thereby exposing a distinctive ensemble of hydrophobic residues to associate specifically with a particular physiological target. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

Effects of Ca2+, Mg2+, and Myristoylation on Guanylyl Cyclase Activating Protein 1 Structure and Stability†

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca(2+) and Mg(2+) binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). GCAP1 binds cooperatively to Ca(2+) at EF3 and EF4 (DeltaH(EF3) = -3.5 kcal/mol, and DeltaH(EF4) = -0.9 kcal/mol) with nanomolar affinity (K(EF3) = 80 nM, and K(EF4) = 200 nM), and a third Ca(2+) binds entropically at EF2 (DeltaH(EF2) = 3.1 kcal/mol, and K(EF2) = 0.9 microM). GCAP1 binds functionally to Mg(2+) at EF2 (DeltaH(EF2) = 4.3 kcal/mol, and K(EF2) = 0.7 mM) required for RetGC activation. Ca(2+) and/or Mg(2+) binding to GCAP1 dramatically alters DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3, and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange-broadened, upfield-shifted, and insensitive to Ca(2+), consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg(2+) or Ca(2+) binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.

Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP

Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H2 utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H2 uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H2 uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H2 metabolism.

Structure of a Ca2+-Myristoyl Switch Protein That Controls Activation of a Phosphatidylinositol 4-Kinase in Fission Yeast

Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins.

Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

Background: N-Myristoylated GCAP1 controls Ca2+ sensitivity of cGMP synthesis in photoreceptors. Results: Hydrophobic residues replacing the myristoyl group in GCAP1 restore activation of its target enzyme but not Ca2+ binding. Conclusion: The fatty chain regulates sensitivity of EF-hand located on the opposite side via intramolecular tug. Significance: Mechanisms of Ca2+ binding by GCAP1 are critical for normal vision and visual disorders. Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca2+ sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca2+ sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca2+ binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca2+-bound and Mg2+-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca2+ sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca2+ binding to EF-hand 4 and Ca2+ sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu80 and Val180) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or “tug”) between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca2+ sensitivity.

Molecular structure and target recognition of neuronal calcium sensor proteins.

Neuronal calcium sensor (NCS) proteins, a sub-branch of the EF-hand superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulates synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression. Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase (RK), NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. We propose that N-terminal myristoylation is critical for shaping each NCS family member into a different structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues that interact with a particular physiological target.

Structural diversity of neuronal calcium sensor proteins and insights for activation of retinal guanylyl cyclase by GCAP1

Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca2+-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca2+-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca2+-binding to both recoverin and NCS-1 cause large protein conformational changes that ejects the covalently attached myristoyl group into the solvent exterior and promotes membrane targeting (Ca2+-myristoyl switch). The GCAP proteins undergo much smaller Ca2+-induced conformational changes and do not possess a Ca2+-myristoyl switch. Recent structures of GCAP1 in both its activator and Ca2+-bound inhibitory states will be discussed to understand structural determinants that control their Ca2+-dependent activation of retinal guanylyl cyclases.

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Protonated Bilin Ring System in Both Photostates.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both CBCRs and phytochromes use photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties, the dark state and the photoproduct. The isolated CBCR domain NpR6012g4 from Nostoc punctiforme is a well-characterized member of the canonical red/green CBCR subfamily, photosensory domains that can function as sensors for light color or intensity to regulate phototactic responses of filamentous cyanobacteria. Such red/green CBCRs utilize conserved Phe residues to tune the photoproduct for green light absorption, but conflicting interpretations of the photoproduct chromophore structure have been proposed. In the hydration model, the proposed photoproduct state is extensively solvated, with a loosely bound, conformationally flexible chromophore. In the trapped-twist model, the photoproduct chromophore is sterically constrained by hydrophobic amino acids, including the known Phe residues. Here, we have characterized chromophore structure in NpR6012g4 using solution nuclear magnetic resonance spectroscopy and a series of labeled chromophores. Four NH resonances are assigned for both the red-absorbing dark state and the green-absorbing photoproduct. Moreover, observed (13)C chemical shifts are in good agreement with those obtained for protonated rather than deprotonated bilins in ab initio calculations. Our results demonstrate that NpR6012g4 has a protonated, cationic bilin π system in both photostates, consistent with a photoproduct structure in which the chromophore is not extensively hydrated.

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-E,anti Chromophore in the Photoproduct.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹⁵N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.