More Like this

1. 1H, 13C, 15N resonance assignments and secondary structure of yeast oligosaccharyltransferase subunit Ost4 and its functionally important mutant Ost4V23D

   https://doi.org/10.1007/s12104-020-09946-7  

   Chaudhary, Bharat P. ; Zoetewey, David ; Mohanty, Smita ( April 2020 , Biomolecular NMR Assignments)

   Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction that occurs in the endoplasmic reticulum of cells during protein synthesis at the ribosome. In the central reaction, a pre-assembled high- mannose sugar is transferred from a lipid-linked donor substrate to the side-chain of an asparagine residue in an -N-X-T/S- sequence (where X is any residue except Proline). This reaction is carried by a membrane-bound multi-subunit enzyme complex, Oligosaccharyltransferase (OST). In humans, genetic defects in OST lead to a group of rare metabolic diseases collectively known as congenital disorders of glycosylation (CDG). Certain mutations are lethal for all organisms. In yeast, the OST is composed of nine non-identical protein subunits. The functional enzyme complex contains eight subunits with either Ost3 or Ost6 at any given time. Ost4, an unusually small protein, plays a very important role in the stabilization of the OST complex. It bridges the catalytic subunit Stt3 with Ost3 (or Ost6) in the Stt3-Ost4-Ost3 (or Ost6) sub-complex. Mutation of any residue from M18-I24 in the trans-membrane helix of yeast Ost4 negatively impacts N-linked glycosylation and the growth of yeast. Indeed, mutation of valine23 to an aspartate impairs OST function in vivo resulting in a lethal phenotype in yeast. To understand the structural mechanism of Ost4 in the stabilization of the enzyme complex, we have initiated a detailed investigation of Ost4 and its functionally important mutant, Ost4V23D. Here, we report the backbone 1H, 13C and 15N resonance assignments for Ost4 and Ost4V23D in DPC micelles. 

   more » « less
2. Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

   https://doi.org/10.3390/biom10040624  

   Mohanty, Smita ; P Chaudhary, Bharat ; Zoetewey, David ( April 2020 , Biomolecules)

   Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal. 

   more » « less
3. Structural and mechanistic studies of the N -glycosylation machinery: from lipid-linked oligosaccharide biosynthesis to glycan transfer

   https://doi.org/10.1093/glycob/cwad053  

   Ramírez, Ana S. ; Locher, Kaspar P. ( July 2023 , Glycobiology)

   N-linked protein glycosylation is a post-translational modification that exists in all domains of life. It involves two consecutive steps: (i) biosynthesis of a lipid-linked oligosaccharide (LLO), and (ii) glycan transfer from the LLO to asparagine residues in secretory proteins, which is catalyzed by the integral membrane enzyme oligosaccharyltransferase (OST). In the last decade, structural and functional studies of the N-glycosylation machinery have increased our mechanistic understanding of the pathway. The structures of bacterial and eukaryotic glycosyltransferases involved in LLO elongation provided an insight into the mechanism of LLO biosynthesis, whereas structures of OST enzymes revealed the molecular basis of sequon recognition and catalysis. In this review, we will discuss approaches used and insight obtained from these studies with a special emphasis on the design and preparation of substrate analogs.

    

   more » « less
4. Novel phosphorylation‐dependent regulation in an unstructured protein

   https://doi.org/10.1002/prot.25812  

   Cannon, John F. ( October 2019 , Proteins: Structure, Function, and Bioinformatics)

   This work explores how phosphorylation of an unstructured protein region in inhibitor‐2 (I2) regulates protein phosphatase‐1 (PP1) enzyme activity using molecular dynamics (MD). Free I2 is largely unstructured; however, when bound to PP1, three segments adopt a stable structure. In particular, an I2 helix (i‐helix) blocks the PP1 active site and inhibits phosphatase activity. I2 phosphorylation in the PP1‐I2 complex activates phosphatase activity without I2 dissociation. The I2 Thr74 regulatory phosphorylation site is in an unstructured domain in PP1‐I2. PP1‐I2 MD demonstrated that I2 phosphorylation promotes early steps of PP1‐I2 activation in explicit solvent models. Moreover, phosphorylation‐dependent activation occurred in PP1‐I2 complexes derived from I2 orthologs with diverse sequences from human, yeast, worm, and protozoa. This system allowed exploration of features of the 73‐residue unstructured human I2 domain critical for phosphorylation‐dependent activation. These studies revealed that components of I2 unstructured domain are strategically positioned for phosphorylation responsiveness including a transient α‐helix. There was no evidence that electrostatic interactions of I2 phosphothreonine74 influenced PP1‐I2 activation. Instead, phosphorylation altered the conformation of residues around Thr74. Phosphorylation uncurled the distance between I2 residues Glu71 to Tyr76 to promote PP1‐I2 activation, whereas reduced distances reduced activation. This I2 residue Glu71 to Tyr76 distance distribution, independently from Thr74 phosphorylation, controls I2 i‐helix displacement from the PP1 active site leading to PP1‐I2 activation.

    

   more » « less
5. A Case Study of the Glycoside Hydrolase Enzyme Mechanism Using an Automated QM-Cluster Model Building Toolkit

   https://doi.org/10.3389/fchem.2022.854318  

   Cheng, Qianyi ; DeYonker, Nathan John ( March 2022 , Frontiers in Chemistry)

   Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol −1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol −1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme. 

   more » « less