Publications by authors named "Meredith L Jenkins"

19 Publications

  • Page 1 of 1

Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex.

J Mol Biol 2021 Jul 3;433(18):167145. Epub 2021 Jul 3.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada; Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address:

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.
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http://dx.doi.org/10.1016/j.jmb.2021.167145DOI Listing
July 2021

The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity.

Commun Biol 2020 12 4;3(1):735. Epub 2020 Dec 4.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.
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http://dx.doi.org/10.1038/s42003-020-01459-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719173PMC
December 2020

and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

J Am Soc Mass Spectrom 2020 Oct 25;31(10):2202-2209. Epub 2020 Sep 25.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for . For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.
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http://dx.doi.org/10.1021/jasms.0c00283DOI Listing
October 2020

Activation of Phospholipase C β by Gβγ and Gα Involves C-Terminal Rearrangement to Release Autoinhibition.

Structure 2020 07 12;28(7):810-819.e5. Epub 2020 May 12.

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address:

Phospholipase C (PLC) enzymes hydrolyze phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gα and/or Gβγ subunits mediates signaling by Gq and some Gi coupled G-protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTDs) separated by a flexible linker. The structure of PLCβ3 bound to Gα is known, however, for both Gα and Gβγ; the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gα showed decreased deuterium incorporation at the Gα binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of autoinhibitory interactions with the CTD leads to increased PLCβ hydrolase activity.
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http://dx.doi.org/10.1016/j.str.2020.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891876PMC
July 2020

Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication.

EMBO Rep 2020 02 12;21(2):e48441. Epub 2019 Dec 12.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.
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http://dx.doi.org/10.15252/embr.201948441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001497PMC
February 2020

Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5.

Nat Commun 2018 09 14;9(1):3772. Epub 2018 Sep 14.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.

The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab-GEF interaction. Furthermore, it reveals a rearrangement of the switch I region of Rab11 compared with solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutation of switch I provides insights into the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases present on Rab11-positive organelles. Moreover, we show that GEF-deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. Overall, our results give molecular insight into Rab11 regulation, and how Rab-GEF specificity is achieved.
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http://dx.doi.org/10.1038/s41467-018-06196-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138693PMC
September 2018

An intrinsic lipid-binding interface controls sphingosine kinase 1 function.

J Lipid Res 2018 03 11;59(3):462-474. Epub 2018 Jan 11.

Department of Medicine and Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY 11790

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.
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http://dx.doi.org/10.1194/jlr.M081307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832922PMC
March 2018

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange.

J Chem Inf Model 2018 02 31;58(2):464-471. Epub 2018 Jan 31.

Department of Organic Chemistry, The Weizmann Institute of Science , Rehovot, 7610001, Israel.

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-Ras. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-Ras. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.
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http://dx.doi.org/10.1021/acs.jcim.7b00399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179444PMC
February 2018

Ras Binder Induces a Modified Switch-II Pocket in GTP and GDP States.

Cell Chem Biol 2017 12 12;24(12):1455-1466.e14. Epub 2017 Oct 12.

Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA. Electronic address:

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.
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http://dx.doi.org/10.1016/j.chembiol.2017.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915340PMC
December 2017

Dissecting the molecular assembly of the MyoA motility complex.

J Biol Chem 2017 11 25;292(47):19469-19477. Epub 2017 Sep 25.

From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada and

Apicomplexan parasites such as rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.
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http://dx.doi.org/10.1074/jbc.M117.809632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702683PMC
November 2017

An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery.

Expert Opin Drug Discov 2017 10 17;12(10):981-994. Epub 2017 Aug 17.

b Department of Biochemistry and Microbiology , University of Victoria , Victoria , Canada.

Introduction: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule interactions. The development of novel methodologies and technical advancements in mass spectrometers has greatly expanded the accessibility and acceptance of this technique within both academia and industry. Areas covered: This review examines the theoretical basis of how amide exchange occurs, how different mass spectrometer approaches can be used for HDX-MS experiments, as well as the use of HDX-MS in drug development, specifically focusing on how HDX-MS is used to characterize bio-therapeutics, and its use in examining protein-protein and protein small molecule interactions. Expert opinion: HDX-MS has been widely accepted within the pharmaceutical industry for the characterization of bio-therapeutics as well as in the mapping of antibody drug epitopes. However, there is room for this technique to be more widely used in the drug discovery process. This is particularly true in the use of HDX-MS as a complement to other high-resolution structural approaches, as well as in the development of small molecule therapeutics that can target both active-site and allosteric binding sites.
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http://dx.doi.org/10.1080/17460441.2017.1363734DOI Listing
October 2017

Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes.

Biochemistry 2017 06 16;56(25):3178-3183. Epub 2017 Jun 16.

Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco , San Francisco, California 94158, United States.

There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compounds to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallography, hydrogen/deuterium exchange, and differential scanning fluorimetry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation.
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http://dx.doi.org/10.1021/acs.biochem.7b00271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5665167PMC
June 2017

Molecular mechanism of activation of class IA phosphoinositide 3-kinases (PI3Ks) by membrane-localized HRas.

J Biol Chem 2017 07 17;292(29):12256-12266. Epub 2017 May 17.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada. Electronic address:

Class IA PI3Ks are involved in the generation of the key lipid signaling molecule phosphatidylinositol 3,4,5-trisphosphate (PIP), and inappropriate activation of this pathway is implicated in a multitude of human diseases, including cancer, inflammation, and primary immunodeficiencies. Class IA PI3Ks are activated downstream of the Ras superfamily of GTPases, and Ras-PI3K interaction plays a key role in promoting tumor formation and maintenance in Ras-driven tumors. Investigating the detailed molecular events in the Ras-PI3K interaction has been challenging because it occurs on a membrane surface. Here, using maleimide-functionalized lipid vesicles, we successfully generated membrane-resident HRas and evaluated its effect on PI3K signaling in lipid kinase assays and through analysis with hydrogen-deuterium exchange MS. We screened all class IA PI3K isoforms and found that HRas activates both p110α and p110δ isoforms but does not activate p110β. The p110α and p110δ activation by Ras was synergistic with activation by a soluble phosphopeptide derived from receptor tyrosine kinases. Hydrogen-deuterium exchange MS revealed that membrane-resident HRas, but not soluble HRas, enhances conformational changes associated with membrane binding by increasing membrane recruitment of both p110α and p110δ. Together, these results afford detailed molecular insight into the Ras-PI3K signaling complex, provide a framework for screening Ras inhibitors, and shed light on the isoform specificity of Ras-PI3K interactions in a native membrane context.
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http://dx.doi.org/10.1074/jbc.M117.789263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519374PMC
July 2017

Conformational disruption of PI3Kδ regulation by immunodeficiency mutations in and .

Proc Natl Acad Sci U S A 2017 02 6;114(8):1982-1987. Epub 2017 Feb 6.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada V8W 2Y2;

Activated PI3K Delta Syndrome (APDS) is a primary immunodeficiency disease caused by activating mutations in either the leukocyte-restricted p110δ catalytic () subunit or the ubiquitously expressed p85α regulatory () subunit of class IA phosphoinositide 3-kinases (PI3Ks). There are two classes of APDS: APDS1 that arises from p110δ mutations that are analogous to oncogenic mutations found in the broadly expressed p110α subunit and APDS2 that occurs from a splice mutation resulting in p85α with a central deletion (Δ434-475). As p85 regulatory subunits associate with and inhibit all class IA catalytic subunits, APDS2 mutations are expected to similarly activate p110α, β, and δ, yet APDS2 largely phenocopies APDS1 without dramatic effects outside the immune system. We have examined the molecular mechanism of activation of both classes of APDS mutations using a combination of biochemical assays and hydrogen-deuterium exchange mass spectrometry. Intriguingly, we find that an APDS2 mutation in p85α leads to substantial basal activation of p110δ (>300-fold) and disrupts inhibitory interactions from the nSH2, iSH2, and cSH2 domains of p85, whereas p110α is only minimally basally activated (∼2-fold) when associated with mutated p85α. APDS1 mutations in p110δ (N334K, E525K, E1021K) mimic the activation mechanisms previously discovered for oncogenic mutations in p110α. All APDS mutations were potently inhibited by the Food and Drug Administration-approved p110δ inhibitor idelalisib. Our results define the molecular basis of how and mutations result in APDS and reveal a potential path to treatment for all APDS patients.
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http://dx.doi.org/10.1073/pnas.1617244114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338455PMC
February 2017

Recognition of protein-linked glycans as a determinant of peptidase activity.

Proc Natl Acad Sci U S A 2017 01 17;114(5):E679-E688. Epub 2017 Jan 17.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6, Canada;

The vast majority of proteins are posttranslationally altered, with the addition of covalently linked sugars (glycosylation) being one of the most abundant modifications. However, despite the hydrolysis of protein peptide bonds by peptidases being a process essential to all life on Earth, the fundamental details of how peptidases accommodate posttranslational modifications, including glycosylation, has not been addressed. Through biochemical analyses and X-ray crystallographic structures we show that to hydrolyze their substrates, three structurally related metallopeptidases require the specific recognition of O-linked glycan modifications via carbohydrate-specific subsites immediately adjacent to their peptidase catalytic machinery. The three peptidases showed selectivity for different glycans, revealing protein-specific adaptations to particular glycan modifications, yet always cleaved the peptide bond immediately preceding the glycosylated residue. This insight builds upon the paradigm of how peptidases recognize substrates and provides a molecular understanding of glycoprotein degradation.
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http://dx.doi.org/10.1073/pnas.1615141114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293097PMC
January 2017

The Molecular Basis of Aichi Virus 3A Protein Activation of Phosphatidylinositol 4 Kinase IIIβ, PI4KB, through ACBD3.

Structure 2017 01 15;25(1):121-131. Epub 2016 Dec 15.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada. Electronic address:

Phosphatidylinositol 4-kinase III beta (PI4KIIIβ) is an essential enzyme in mediating membrane transport, and plays key roles in facilitating viral infection. Many pathogenic positive-sense single-stranded RNA viruses activate PI4KIIIβ to generate phosphatidylinositol 4-phosphate (PI4P)-enriched organelles for viral replication. The molecular basis for PI4KIIIβ activation during viral infection has remained largely unclear. We describe the biochemical reconstitution and characterization of the complex of PI4KIIIβ with the Golgi protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3) and Aichi virus 3A protein on membranes. We find that 3A directly activates PI4KIIIβ, and this activation is sensitized by ACBD3. The interfaces between PI4KIIIβ-ACBD3 and ACBD3-3A were mapped with hydrogen-deuterium exchange mass spectrometry (HDX-MS). Determination of the crystal structure of the ACBD3 GOLD domain revealed a unique N terminus that mediates the interaction with 3A. Rationally designed complex-disrupting mutations in both ACBD3 and PI4KIIIβ completely abrogated the sensitization of 3A activation by ACBD3.
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http://dx.doi.org/10.1016/j.str.2016.11.016DOI Listing
January 2017

Molecular Basis for Recognition of the Cancer Glycobiomarker, LacdiNAc (GalNAc[β1→4]GlcNAc), by Wisteria floribunda Agglutinin.

J Biol Chem 2016 Nov 6;291(46):24085-24095. Epub 2016 Sep 6.

From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 3P6, Canada and

Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding K values of 4.67 × 10 m, 9.24 × 10 m, and 5.45 × 10 m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80-2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen).
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http://dx.doi.org/10.1074/jbc.M116.750463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104934PMC
November 2016

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11.

Protein Sci 2016 Apr 1;25(4):826-39. Epub 2016 Feb 1.

Department of Biochemistry and Microbiology, University of Victoria, British Columbia, V8P 5C2, Canada.

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches.
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http://dx.doi.org/10.1002/pro.2879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832280PMC
April 2016
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