Chi Ma, FuKun W. Hoffmann, Michael P. Marciel, Kathleen E. Page, Melodie A. Williams-Aduja, Ellis N. L. Akana, Greg S. Gojanovich, Mariana Gerschenson, Johann Urschitz, Stefan MoisyadiVedbar S. KhadkaSharon Rozovsky, Youping Deng, F. David Horgen, and Peter R. Hoffmann Upregulated ethanolamine phospholipid synthesis by selenoprotein I is required for effective metabolic reprogramming during T cell activationIn press Molecular Metabolism 

Abstract

Objective: T cell activation triggers metabolic reprogramming to meet increased demand for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cell and cancer cells, which led us to investigate a potential role during T cell activation.

Methods: Since selenoprotein I (SELENOI) is an enzyme participating in two synthesis pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. Results: SELENOI knockout (KO) in mouse T cells exhibited reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor, AMPK. AMPK is inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment. Conclusions: T cell activation upregulates SELENOIdependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.

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Jun Liu, Lingchao Cai, Wei Sun, Rujin Cheng, Nanxi Wang, Ling Jin, Sharon Rozovsky, Ian Seiple, and Lei WangPhotocaged Quinone Methide Cross-linkers for Light-controlled Chemical Cross-linking of Protein-protein and Protein-DNA complexes. Angew. Chem. Int. Ed. (2020) 

Abstract

Small molecule cross-linkers are invaluable for probing biomolecular interactions and for cross-linking mass spectrometry (CXMS) in addressing large protein complexes and intrinsically disordered proteins. Existing chemical cross-linkers target only a small selection of amino acid residues, limiting the number and type of cross-links, while conventional photocross-linkers target virtually all residues non-selectively, complicating data analysis. Here we report photocaged quinone methide (PQM)-based cross-linkers that are able to multitarget nine nucleophilic residues through specific Michael addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM crosslinkers notably cross-linked Gln, Arg, and Asn hitherto untargetable by existing chemical cross-linkers, markedly increasing the number of residues targetable with a single cross-linker. Such multiplicity of cross-links will increase the abundance of cross-linked peptides for CXMS identification and afford ample constraints to facilitate structural deciphering. PQM cross-linkers were used in vitro, in E. coli, and in mammalian cells to cross-link dimeric proteins and endogenous membrane receptors. The cross-linker NHQM could directly cross-link proteins to DNA, for which few cross-linkers exist. The photoactivatable and multitargeting reactivity of these PQM cross-linkers will substantially enhance chemical cross-linking based technologies for studies of protein-protein and protein-DNA networks and for structural biology.

Link to Article

Rujin Cheng, Jun Liu, Wang Lei, Martin Forstner and Sharon RozovskyChemical biological synthesis of Selenoproteins. In “Encyclopedia of Biological Chemistry”, 3rd Edition (2020) Edited by Norma Allwell.

Jun Liu, Rujin Cheng, Ned Van Eps, Nanxi Wang, Takefumi Morizumi, Wei-Lin Ou, Paul Klauser, Sharon Rozovsky, Oliver Ernst, and Lei Wang. Genetically Encoded Quinone Methides Enabling Rapid, Site-specific, and Photo-controlled Protein Modification with Amine Reagents. Journal of the American Chemical Society,142, 40, 17057-17068 (2020)

Abstract

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the β-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron–electron paramagnetic resonance distance measurements.

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Qingqing Chen, Shiping Xu, Xingyu Lu, Michael Boeri, Yuliya Pepelyayeva, Elizabeth Diaz, Sunil-Datta Soni, Marc Allaire, Martin Forstner, Brian Bahnson, and Sharon Rozovsky77Se-NMR probes the protein environment of selenomethionine. Journal of Physical Chemistry B (2019) 

Abstract

Sulfur is critical for the correct structure and proper function of proteins. Yet, lacking a sensitive enough isotope, nuclear magnetic resonance (NMR) experiments are unable to deliver for sulfur in proteins the usual wealth of chemical, dynamic and structural information. This limitation can be circumvented by substituting sulfur with selenium, which has similar physicochemical properties and minimal impact on protein structures but possesses an NMR compatible isotope (77Se). Here we exploit the sensitivity of 77Se-NMR to the nucleus’ chemical milieu and use selenomethionine as a probe for its proteinaceous environment. However, such selenium NMR spectra of proteins currently resist a reliable interpretation because systematic connections between variations of system variables and changes in 77Se-NMR parameters are still lacking. To start narrowing this knowledge gap we report here on a biological 77Se magnetic resonance databank based on a systematically designed library of GB1 variants in which a single selenomethionine was introduced at different locations within the protein. We recorded the resulting isotropic 77Se chemical shifts and relaxation times for six GB1 variants by solution 77Se-NMR. For four of the GB1 variants we were also able to determine the chemical shift anisotropy tensor of SeM by solid-state 77Se-NMR. To enable interpretation of the NMR data, the structures of five of the GB1 variants were solved by x-ray crystallography to a resolution of 1.2 Å, allowing us to unambiguously determine the conformation of the selenomethionine. Finally, we combine our solution and solid-state NMR data with the structural information to arrive at general insights regarding the execution and interpretation of 77Se-NMR experiments that exploit selenomethionine to probe proteins.

 

Link to Article

Jun Liu, Oshini Ekanayake, Dominic Santoleri, Kelsi Walker, and Sharon Rozovsky. Efficient generation of hydrazides in proteins by RadA split intein. ChemBioChem (2019) 

Abstract

Proteins’ C-terminal hydrazides are useful for bioconjugation and constructing proteins from multiple fragments via native chemical ligation. To improve the preparation of C-terminal hydrazides, we developed an efficient intein-based preparation method using thiols and hydrazine to accelerate formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield but also improves biocompatibility. We expanded the method’s versatility by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. Besides, the use of split RadA helped prevent intein proteolytic cleavage in vivo. It also offers greater control over initiation of the intein cleavage reaction. We demonstrate the preparation of C-terminal hydrazide in model proteins as well as its conjugation with an aldehyde.

Samuel L. Scinto, Oshini Ekanayake, Uthpala Seneviratne, Jessica Pigga, Samantha J. Boyd, Michael T. Taylor, Jun Liu, Christopher W. am Ende, Sharon Rozovsky, Joe Fox. Dual reactivity trans-cyclooctenol probes for sulfenylation in live cells enable temporal control via bioorthogonal quenching. JACS (2019) 

Abstract

Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas “ordinary” trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

Jun Liu, Shanshan Li, Nayyar A. Aslam, Feng Zheng, Bing Yang, Rujin Cheng, Nanxi Wang, Sharon Rozovsky, Peng G. Wang, Qian Wang, and Lei Wang. Genetically encoding photocaged quinone methide to multitarget protein residues covalently in vivo JACS (2019) 

Abstract

Genetically introducing covalent bonds into proteins in vivo with residue specificity is affording innovative ways for protein research and engineering, yet latent bioreactive unnatural amino acids (Uaas) genetically encoded to date react with one to few natural residues only, limiting the variety of proteins and the scope of applications amenable to this technology. Here we report the genetic encoding of (2R)-2-amino-3-fluoro-3-(4-((2-nitrobenzyl)oxy) phenyl) propanoic acid (FnbY) in Escherichia coli and mammalian cells. Upon photoactivation, FnbY generated a reactive quinone methide (QM), which selectively reacted with nine natural amino acid residues placed in proximity in proteins directly in live cells. In addition to Cys, Lys, His, and Tyr, photoactivated FnbY also reacted with Trp, Met, Arg, Asn, and Gln, which are inaccessible with existing latent bioreactive Uaas. FnbY thus dramatically expanded the number of residues for covalent targeting in vivo. QM has longer half-life than the intermediates of conventional photo-cross-linking Uaas, and FnbY exhibited cross-linking efficiency higher than p-azido-phenylalanine. The photoactivatable and multitargeting reactivity of FnbY with selectivity toward nucleophilic residues will be valuable for addressing diverse proteins and broadening the scope of applications through exploiting covalent bonding in vivo for chemical biology, biotherapeutics, and protein engineering.

Jun Liu, Rujin Cheng, Haifan Wu, Shanshan Li, Peng G. Wang, William F. DeGrado, Sharon Rozovsky, Lei Wang. Build and break bonds via a compact S-propargyl-cysteine to chemically control enzymes and modify proteins. Angewandte Chemie 57(39):12702-12706 (2018).

Abstract

Analogous to reversible post-translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S-propargyl-cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol-yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd-cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.

Liu J, Zheng F, Cheng R, Li S, Rozovsky S, Wang Q, Wang L. Site-specific incorporation of selenocysteine using an expanded genetic code and palladium-mediated chemical deprotection.  JACS 140 (28):8807–8816 (2018).

Abstract

Selenoproteins containing the 21st amino acid selenocysteine (Sec) exist in all three kingdoms of life and play essential roles in human health and development. The distinct low p Ka, high reactivity, and redox property of Sec also afford unique routes to protein modification and engineering. However, natural Sec incorporation requires idiosyncratic translational machineries that are dedicated to Sec and species-dependent, which makes it challenging to recombinantly prepare selenoproteins with high Sec specificity. As a consequence, the function of half of human selenoproteins remains unclear, and Sec-based protein manipulation has been greatly hampered. Here we report a new general method enabling the site-specific incorporation of Sec into proteins in E. coli. An orthogonal tRNAPyl-ASecRS was evolved to specifically incorporate Se-allyl selenocysteine (ASec) in response to the amber codon, and the incorporated ASec was converted to Sec in high efficiency through palladium-mediated cleavage under mild conditions compatible with proteins and cells. This approach completely obviates the natural Sec-dedicated factors, thus allowing various selenoproteins, regardless of Sec position and species source, to be prepared with high Sec specificity and enzyme activity, as shown by the preparation of human thioredoxin and glutathione peroxidase 1. Sec-selective labeling in the presence of Cys was also demonstrated on the surface of live E. coli cells. The tRNAPyl-ASecRS pair was further used in mammalian cells to incorporate ASec, which was converted into Sec by palladium catalyst in cellulo. This robust and versatile method should greatly facilitate the study of diverse natural selenoproteins and the engineering of proteins in general via site-specific introduction of Sec.fications.

Jun Liu, Rujin Cheng and Sharon Rozovsky. Synthesis and semi-synthesis of selenopeptides and selenoproteins. Current Opinion in Chemical Biology, 46, 41-47 (2018).

Abstract

The versatile chemistry of the genetically encoded amino acid selenocysteine (Sec) is employed in Nature to expand the reactivity of enzymes. In addition to, its role in biology, Sec is used in protein engineering to modify folding, stability, and reactivity of proteins, to introduce conjugations and to facilitate reactions. However, due to limitations related to Sec’s insertion mechanism in Nature, much of the production of Sec containing peptides and proteins relies on synthesis and semisynthesis. Here, we review recent advances that have enabled the assembly of complicated selenoproteins, including novel uses of protecting groups for solid phase peptide synthesis, rapid selenoester driven chemical ligations and versatile expressed protein ligations.

Clara S. Chan, Sean M. McAllister, Arkadiy Garber, Beverly J. Hallahan, and Sharon Rozovsky. Fe oxidation by a fused cytochrome-porin common to diverse Fe-oxidizing bacteria.  Submitted (2018) Posted on bioRxiv, 228056

Abstract

Fe oxidation is one of Earths major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our ability to track the contributions of Fe-oxidizing microbes is limited by our relatively incomplete knowledge of microbial Fe oxidation mechanisms, particularly in neutrophilic Fe-oxidizers. The genomes of many Fe-oxidizers encode homologs to an outer-membrane cytochrome (Cyc2) that has been shown to oxidize Fe in two acidophiles. Here, we demonstrate the Fe oxidase function of a heterologously expressed Cyc2 homolog derived from a neutrophilic Fe oxidizer. Phylogenetic analyses show that Cyc2 from neutrophiles cluster together, suggesting a common function. Sequence analysis and modeling reveal the entire Cyc2 family is defined by a unique structure, a fused cytochrome-porin, consistent with Fe oxidation on the outer membrane, preventing internal Fe oxide encrustation. Metatranscriptomes from Fe-oxidizing environments show exceptionally high expression of cyc2, supporting its environmental role in Fe oxidation. Together, these results provide evidence that cyc2 encodes Fe oxidases in diverse Fe-oxidizers and therefore can be used to recognize microbial Fe oxidation. The presence of cyc2 in 897 genomes suggests that microbial Fe oxidation may be a widespread metabolism.

Gregory J. Fredericks, FuKun W. Hoffmann, Robert J. Hondal, Sharon Rozovsky, Johann Urschitz, Peter R. Hoffmann. Selenoprotein K increases efficiency of DHHC6 catalyzed protein palmitoylation by stabilizing the acyl-DHHC6 intermediate.  Antioxidants 7(1), 4 (2018).

Abstract

Selenoprotein K (SELENOK) is a selenocysteine (Sec)-containing protein localized in the endoplasmic reticulum (ER) membrane where it interacts with the DHHC6 (where single letter symbols represent Asp-His-His-Cys amino acids) enzyme to promote protein acyl transferase (PAT) reactions. PAT reactions involve the DHHC enzymatic capture of palmitate via a thioester bond to cysteine (Cys) residues that form an unstable palmitoyl-DHHC intermediate, followed by transfer of palmitate to Cys residues of target proteins. How SELENOK facilitates this reaction has not been determined. Splenocyte microsomal preparations from wild-type mice versus SELENOK knockout mice were used to establish PAT assays and showed decreased PAT activity (~50%) under conditions of SELENOK deficiency. Using recombinant, soluble versions of DHHC6 along with SELENOK containing Sec92, Cys92, or alanine (Ala92), we evaluated the stability of the acyl-DHHC6 intermediate and its capacity to transfer the palmitate residue to Cys residues on target peptides. Versions of SELENOK containing either Ala or Cys residues in place of Sec were equivalently less effective than Sec at stabilizing the acyl-DHHC6 intermediate or promoting PAT activity. These data suggest that Sec92 in SELENOK serves to stabilize the palmitoyl-DHHC6 intermediate by reducing hydrolyzation of the thioester bond until transfer of the palmitoyl group to the Cys residue on the target protein can occur.

Aaron J. Wolfe, Wei Si, Zhengqi Zhang, Adam R. Blanden, Yi-Ching Hsueh, Jack F. Gugel, Bach Pham, Min Chen, Stewart N. Loh, Sharon Rozovsky, Aleksei Aksimentiev, and Liviu Movileanu. Quantification of membrane protein-detergent complex interactions. Journal of Physical Chemistry B, 121 (44), pp 10228–10241 (2017).

Abstract

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

Qi Chen, Sharon Rozovsky, and Wilfred Chen. Engineering multi-functional bacterial outer membrane vesicles as modular nanodevices for biosensing and bioimaging.  ChemComm 53(54):7569-7572 (2017)

Abstract

Outer membrane vesicles (OMVs) are proteoliposomes derived from the outer membrane and periplasmic space of many Gram-negative bacteria including E. coli as part of their natural growth cycle. Inspired by the natural ability of E. coli to sort proteins to both the exterior and interior of OMVs, we reported here a one-pot synthesis approach to engineer multi-functionalized OMV-based sensors for both antigen binding and signal generation. SlyB, a native lipoprotein, was used a fusion partner to package nanoluciferase (Nluc) within OMVs, while a previously developed INP-Scaf3 surface scaffold was fused to the Z-domain for antibody recruiting. The multi-functionalized OMVs were used for thrombin detection with a detection limit of 0.5 nM, comparable to other detection methods. Using the cohesin domains inserted between the Z-domain and INP, these engineered OMVs were further functionalized with a dockerin-tagged GFP for cancer cell imaging.

Jun Liu, Qingqing Chen and Sharon Rozovsky. Selenocysteine mediated expressed protein ligation of SELENOM. In “Selenoproteins: Methods and Protocols, Methods in Molecular Biology”, Edited by L. Chavattee, Vol. 1661, pages 265:283 (2017)

Abstract

A sizeable fraction of the selenoproteome encodes oxidoreductases possessing a thioredoxin fold, a structural motif that is shared among a diverse group of enzymes. In these oxidoreductases, the active site is comprised of a cysteine and a selenocysteine separated by one to two amino acids. In a subset of these selenoproteins, such as human SELENOH, SELENOM, SELENOT, SELENOV, SELENOW, and SELENOF, this redox motif is positioned immediately after the first β-sheet in a short loop, and is essential for interactions with its substrate or partners. Here, we describe the preparation of a representative member of this group, SELENOM, by selenocysteine-driven expressed protein ligation. The preparation employs a peptide bond formation between two protein fragments expressed recombinantly in E. coli. This method can be employed to prepare other selenoproteins.

Zhengqi Zhang, Jun Liu, and Sharon Rozovsky. Preparation of selenocysteine-containing forms of human SELENOK and SELENOS. In “Selenoproteins: Methods and Protocols, Methods in Molecular Biology”, Edited by L. Chavattee.   Vol. 1661, pages 241:263 (2017)

Abstract

Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmic-reticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Sec-containing forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli’s native Sec incorporation mechanism.

Jun Liu, Qingqing Chen and Sharon Rozovsky. Utilizing selenocysteine for expressed protein ligation and bioconjugation. JACS 139(9):3430-3437 (2017)

Abstract

Employing selenocysteine-containing protein fragments to form the amide bond between respective protein fragments significantly extends the current capabilities of the widely used protein engineering method, expressed protein ligation. Selenocysteine-mediated ligation is noteworthy for its high yield and efficiency. However, it has so far been restricted to solid-phase synthesized seleno-peptides and thus constrained by where the selenocysteine can be positioned. Here we employ heterologously expressed seleno-fragments to overcome the placement and size restrictions in selenocysteine-mediated chemical ligation. Following ligation, the selenocysteine can be deselenized into an alanine or serine, resulting in nonselenoproteins. This greatly extends the flexibility in selecting the conjugation site in expressed protein ligations with no influence on native cysteines. Furthermore, the selenocysteine can be used to selectively introduce site-specific protein modifications. Therefore, selenocysteine-mediated expressed protein ligation simplifies incorporation of post-translational modifications into the protein scaffold.

Sai Kalburge, Megan Carpenter, Sharon Rozovsky, and Ethna Boyd. Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus. Infection and Immunity 85(3), e00930-16 (2017)

Abstract

Quorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO is active and regulates the two QS master regulators AphA, which is induced, and OpaR, which is repressed. At high cell density the opposite occurs: LuxO is inactive, and therefore OpaR is induced while AphA is repressed. In Vibrio parahaemolyticus, a significant enteric pathogen of humans, the roles of these regulators in pathogenesis are less known. We examined deletion mutants of luxOopaR, and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to levels in the wild type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO opaR double mutant and was also increased in an opaR aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. Transcriptome sequencing (RNA-Seq) analysis of the wild type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.

Gladyshev, V. N.; Arner, E. S.; Berry, M. J.; Brigelius-Flohe, R.; Bruford, E. A.; Burk, R. F.; Carlson, B. A.; Castellano, S.; Chavatte, L.; Conrad, M.; Copeland, P. R.; Diamond, A. M.; Driscoll, D. M.; Ferreiro, A.; Flohe, L.; Green, F. R.; Guigo, R.; Handy, D. E.; Hatfield, D. L.; Hesketh, J.; Hoffmann, P. R.; Holmgren, A.; Hondal, R. J.; Howard, M. T.; Huang, K.; Kim, H. Y.; Kim, I. Y.; Kohrle, J.; Krol, A.; Kryukov, G. V.; Lee, B. J.; Lee, B. C.; Lei, X. G.; Liu, Q.; Lescure, A.; Lobanov, A. V.; Loscalzo, J.; Maiorino, M.; Mariotti, M.; Prabhu, K. S.; Rayman, M. P.; Rozovsky, S.; Salinas, G.; Schmidt, E. E.; Schomburg, L.; Schweizer, U.; Simonovic, M.; Sunde, R. A.; Tsuji, P. A.; Tweedie, S.; Ursini, F.; Whanger, P. D.; Zhang, Y. Selenoprotein gene nomenclature. J. Biol. Chem. 291(46), 24036-24040 (2016)

Abstract

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Eric Block, Squire Booker, Sonia Flores-Penalba, Graham George, Bradley Landgraf, Jun Liu, Stephene N. Lodge, M. Jake Pushie, Sharon Rozovsky, Abith Vattekkatte, Rama Yaghi, and Huawei Zeng. Trifluoroselenomethionine – a new non-natural amino acid. CHEMBIOCHEM  Volume 17, Issue 18,  1738–1751 (2016)

Abstract

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N‐(tert‐butoxycarbonyl)‐l‐aspartic acid tert‐butyl ester. TFSeM shows enhanced methioninase‐induced cytotoxicity, relative to selenomethionine (SeM), toward HCT‐116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.

Jun Liu and Sharon Rozovsky. 77Se NMR spectroscopy of selenoproteins. In “Selenium Its molecular Biology and Role in Human Health.” Fourth Edition. Edited by D.L. Hatfield, V.N. Gladyshev, U. Schweizer and P.A. Tsuji. Springer, Chapter 15, 187-198 (2016).

Abstract

One of the most essential contributions of selenium to biology is the specialized chemistry performed by selenoproteins. Elucidating the mechanisms by which selenoproteins govern the reactivity of their selenocysteine (Sec) requires exploring how the protein environment primes Sec interactions with substrates, prevents inactivation, and otherwise optimizes the use of this unique amino acid. 77Se nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique to study the chemical properties of selenocysteine, its conformational preferences and mobility, and the molecular interactions by which it is stabilized. Recent advances have simplified sample preparation and data analysis, extending the utilization of 77Se in NMR studies of biological samples. These improvements include the development of efficient procedures for enriching proteins with the 77Se isotope, the reports on NMR parameters of different selenoproteins that greatly expand the available basis for data analysis, and the progress in utilizing theoretical calculations for data interpretation. We discuss these areas of progress in 77Se NMR of biological systems, and we consider the range of questions for which 77Se NMR is most useful.

Megan R. Carpenter, Sharon Rozovsky and E. Fidelma Boyd. Pathogenicity island cross-talk mediated by recombination directionality factors facilitates excision from the chromosome. Journal of Bacteriology 198(5), 766-776 (2015)

Abstract

Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibriopathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the intpromoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs.

This paper was among the Articles of Significant Interest Selected from This Issue by the Editors  – See summary

It also won the 2016 Carsons Best Graduate Student Publication Award

Jun Liu and Sharon Rozovsky. Membrane-bound selenoproteins.  Antioxidant and Redox Signaling 23 (10), 795-813 (2015)

Abstract

Significance: Selenoproteins employ selenium to supplement the chemistry available through the common 20 amino acids. These powerful enzymes are affiliated with redox biology, often in connection with the detection, management, and signaling of oxidative stress. Among them, membrane-bound selenoproteins play prominent roles in signaling pathways, Ca2+ regulation, membrane complexes integrity, and biosynthesis of lipophilic molecules. Recent Advances: The number of selenoproteins whose physiological roles, protein partners, expression, evolution, and biosynthesis are characterized is steadily increasing, thus offering a more nuanced view of this specialized family. This review focuses on human membrane selenoproteins, particularly the five least characterized ones: selenoproteins I, K, N, S, and T. Critical Issues: Membrane-bound selenoproteins are the least understood, as it is challenging to provide the membrane-like environment required for their biochemical and biophysical characterization. Hence, their studies rely mostly on biological rather than structural and biochemical assays. Another aspect that has not received much attention is the particular role that their membrane association plays in their physiological function. Future Directions: Findings cited in this review show that it is possible to infer the structure and the membrane-binding mode of these lesser-studied selenoproteins and design experiments to examine the role of the rare amino acid selenocysteine. Antioxid. Redox Signal. 23, 795–813.

Illustration from this review article was selected as cover art.

Jochem Struppe, Yong Zhang and Sharon Rozovsky. 77Se chemical shift tensor of L-selenocystine: Experimental NMR measurements and quantum chemical investigations of structural effects. Journal of Physical Chemistry B, 119 (9), 3643-3650 (2015). 

Abstract

The genetically encoded amino acid selenocysteine and its dimeric form, selenocystine, are both utilized by nature. They are found in active sites of selenoproteins, enzymes that facilitate a diverse range of reactions, including the detoxification of reactive oxygen species and regulation of redox pathways. Due to selenocysteine and selenocystine’s specialized biological roles, it is of interest to examine their 77Se NMR properties and how those can in turn be employed to study biological systems. We report the solid-state 77Se NMR measurements of the l-selenocystine chemical shift tensor, which provides the first experimental chemical shift tensor information on selenocysteine-containing systems. Quantum chemical calculations of l-selenocystine models were performed to help understand various structural effects on 77Se l-selenocystine’s chemical shift tensor. The effects of protonation state, protein environment, and substituent of selenium-bonded carbon on the isotropic chemical shift were found to be in a range of ca. 10–20 ppm. However, the conformational effect was found to be much larger, spanning ca. 600 ppm for the C–Se–Se–C dihedral angle range of −180° to +180°. Our calculations show that around the minimum energy structure with a C–Se–Se–C dihedral angle of ca. −90°, the energy costs to alter the dihedral angle in the range from −120° to −60° are within only 2.5 kcal/mol. This makes it possible to realize these conformations in a protein or crystal environment. 77Se NMR was found to be a sensitive probe to such changes and has an isotropic chemical shift range of 272 ± 30 ppm for this energetically favorable conformation range. The energy-minimized structures exhibited calculated isotropic shifts that lay within 3–9% of those reported in previous solution NMR studies. The experimental solid-state NMR isotropic chemical shift is near the lower bound of this calculated range for these readily accessible conformations. These results suggest that the dihedral information may be deduced for a protein with appropriate structural models. These first-time experimental and theoretical results will facilitate future NMR studies of selenium-containing compounds and proteins.

Jun Liu, Zhengqi Zhang and Sharon Rozovsky. Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential. FEBS Lett 588, 3311-3321 (2014). 

Abstract

Selenoprotein K (SelK) is a membrane protein involved in antioxidant defense, calcium regulation and the ER‐associated protein degradation pathway. We found that SelK exhibits a peroxidase activity with a rate that is low but within the range of other peroxidases. Notably, SelK reduced hydrophobic substrates, such as phospholipid hydroperoxides, which damage membranes. Thus, SelK might be involved in membrane repair or related pathways. SelK was also found to contain a diselenide bond—the first intramolecular bond of that kind reported for a selenoprotein. The redox potential of SelK was −257 mV, significantly higher than that of diselenide bonds in small molecules or proteins. Consequently, SelK can be reduced by thioredoxin reductase. These finding are essential for understanding SelK activity and function.

Fei Li, Patricia B. Lutz, Yuliya Pepelyayeva, Elias S.J. Arner, Craig A. Bayse and Sharon Rozovsky. Redox active motifs in selenoproteins. Proceedings of the National Academy of Sciences 111 (19), 6976-6981 (2014). 

Abstract

Significance

In redox biology, the chemistry performed by proteins that contain the rare amino acid selenocysteine is frequently critical to the detoxification of reactive species that are harmful to cellular function. Selenocysteine and cysteine partner to form a motif featuring a sulfur–selenium covalent bond in many selenoproteins. This work demonstrates that selenium NMR, when paired with calculations, can provide critical insight concerning the local environment of these enigmatic redox motifs. It details how redox potentials, conformational preferences, and mobilities of such redox motifs change when the local environment of the selenocysteine is varied. Surprisingly, reverting selenocysteine to cysteine exerts only minor effects on redox potential. These new approaches deepen our understanding of the chemical reactivity and thermodynamic properties of selenoenzymes.

Stephanie Schaefer-Ramadan, Colin Thorpe and Sharon Rozovsky. Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein Augmenter of Liver Regeneration. Archives of Biochemistry and Biophysics 548, 60-65 (2014).

Abstract

Augmenter of liver regeneration (sfALR) is a small disulfide-bridged homodimeric flavoprotein with sulfhydryl oxidase activity. Here, we investigate the catalytic and spectroscopic consequences of selectively replacing C145 by a selenocysteine to complement earlier studies in which random substitution of ∼90% of the 6 cysteine residues per sfALR monomer was achieved growing Escherichia coli on selenite. A selenocysteine insertion sequence (SECIS) element was installed within the gene for human sfALR. SecALR2 showed a spectrum comparable to that of wild-type sfALR. The catalytic efficiency of SecALR2 towards dithiothreitol was 6.8-fold lower than a corresponding construct in which position 145 was returned to a cysteine residue while retaining the additional mutations introduced with the SECIS element. This all-cysteine control enzyme formed a mixed disulfide between C142 and β-mercaptoethanol releasing C145 to form a thiolate-flavin charge transfer absorbance band at ∼530 nm. In contrast, SecALR2 showed a prominent long-wavelength absorbance at 585 nm consistent with the expectation that a selenolate would be a better charge-transfer donor to the isoalloxazine ring. These data show the robustness of the ALR protein fold towards the multiple mutations required to insert the SECIS element and provide the first example of a selenolate to flavin charge-transfer complex.

Illustration from this review article was selected as cover art.

Jun Liu and Sharon Rozovsky. The contribution of selenocysteine to the peroxidase activity of selenoprotein S. Biochemistry 52 (33), 5514–5516 (2013).

Abstract

Selenoprotein S (SelS, VIMP) is an intrinsically disordered enzyme that utilizes selenocysteine to catalyze the reduction of disulfide bonds and peroxides. Here it is demonstrated that selenocysteine is the residue oxidized by the peroxide substrate. It is possible to trap the reaction intermediate selenenic acid when the resolving cysteine is mutated. The selenocysteine allows SelS to rapidly re-form its selenenylsulfide bond following its reduction, and to resist inactivation by H2O2. We propose that SelS’s peroxidase mechanism is similar to that of atypical 2-Cys peroxiredoxin and that selenocysteine allows SelS to sustain activity under oxidative stress.

Sharon Rozovsky. “77Se NMR spectroscopy of selenoproteins” Invited review “Biochalcogen Chemistry: The Biological Chemistry of Sulfur, Selenium, and Tellurium” edited by C.A. Bayse and J.L. Brumaghim, ACS press, chapter 6, 127-142 (2013).  

Abstract

Selenium is a cardinal contributor to cellular antioxidative defense by way of selenoproteins, a family of enzymes that contains the reactive amino acid selenocysteine. This unique group of proteins can be studied by 77Se NMR spectroscopy, a versatile spectroscopic method capable of recording the electronic structure of reactive enzymatic centers. 77Se is a spin 1/2 nucleus with a pronounced chemical shielding response that renders it highly sensitive to its local environment. This review discusses the application of 77Se NMR spectroscopy to studies of native and nonnative selenoproteins, sample preparation, and data interpretation. We also discuss perspectives for the routine use of 77Se in investigations of biological systems and the prospects of using selenium as a spectroscopic surrogate for sulfur, an abundant biological nuclei that is not easily detected by NMR spectroscopy.

Jun Liu, Fei Li and Sharon Rozovsky. The intrinsically disordered membrane protein selenoprotein S is a reductase in vitro. Biochemistry 52 (18), 3051–3061 (2013).

Abstract

Selenoprotein S (SelS or VIMP) is an intrinsically disordered membrane enzyme that provides protection against reactive oxidative species. SelS is a member of the endoplasmic reticulum-associated protein degradation pathway, but its precise enzymatic function is unknown. Because it contains the rare amino acid selenocysteine, it belongs to the family of selenoproteins, which are typically oxidoreductases. Its exact enzymatic function is key to understanding how the cell regulates the response to oxidative stress and thus influences human health and aging. To identify its enzymatic function, we have isolated the selenocysteine-containing enzyme by relying on the aggregation of forms that do not have this reactive residue. That allows us to establish that SelS is primarily a thioredoxin-dependent reductase. It is capable of reducing hydrogen peroxide but is not an efficient or broad-spectrum peroxidase. Only the selenocysteine-containing enzyme is active. In addition, the reduction potential of SelS was determined to be −234 mV using electrospray ionization mass spectrometry. This value is consistent with SelS being a partner of thioredoxin. On the basis of this information, SelS can directly combat reactive oxygen species but is also likely to participate in a signaling pathway, via a yet unidentified substrate.

This article was highlighted as a “Key Scientific Articles” in the Global Medical Discovery – a prominent research news website that highlights landmark papers in science and medicine. It was also highlighted on Biochemistry’s home page.
http://globalmedicaldiscovery.com/key-scientific-articles/the-intrinsically-disordered-membrane-protein-selenoprotein-s-is-a-reductase-in-vitro/

Stephanie A. Schaefer, Ming Dong, Renee P. Rubenstein, Wayne A. Wilkie, Brian J. Bahnson, Colin Thorpe and Sharon Rozovsky.  77Se enrichment of proteins expands the biological NMR toolbox. Journal of Molecular Biology 425, 222-231 (2013).

Abstract

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable 77Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate 77Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidaseaugmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, 77Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.

Jun Liu, Prabha Srinivasan, Diane N. Pham and Sharon Rozovsky.  Expression and purification of the membrane enzyme selenoprotein K. Protein Expression and Purification 86 (1), 27–34 (2012)

Abstract

Selenoprotein K (SelK) is a membrane protein residing in the endoplasmic reticulum. The function of SelK is mostly unknown; however, it has been shown to participate in anti-oxidant defense, calcium regulation and in the endoplasmic reticulum associated protein degradation (ERAD) pathway. In order to study the function of SelK and the role of selenocysteine in catalysis, we have tested heterologous expression of human SelK in E. coli. Consequently, we have developed an over-expression strategy that exploits the maltose binding protein as a fusion partner to stabilize and solubilize SelK. The fusion partner can be cleaved from SelK in the presence of a variety of detergents compatible with structural characterization and the protein purified to homogeneity. SelK acquires a helical secondary structure in detergent micelles, even though it was predicted to be an intrinsically disordered protein due to its high percentage of polar residues. The same strategy was successfully applied to preparation of SelK binding partner – selenoprotein S (SelS). Hence, this heterologous expression and purification strategy can be applied to other members of the membrane enzyme family to which SelK belongs.

Rozovsky, S., Forstner, M.B., Sondermann, H. and Groves, J. T.  Single molecule kinetics of ENTH binding to lipid membranes. Journal of Physical Chemistry 116 (17), 5122-5131 (2012).

Abstract

Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins’ localization and interactions. Thus, the specificity and the kinetics of peripheral proteins’ membrane residence are an attribute of their function. Here, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P2 despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. Furthermore, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH’s diffusive transport appears to be the result of at least three different diffusion processes.

Rozovsky, S. & McDermott, A. E. Substrate product equilibrium on a reversible enzyme: NMR detection for triosephosphate isomerase. Proceedings of the National Academy of Sciences 104 (7), 2080-2085 (2007).

Abstract

The highly efficient glycolytic enzyme, triosephosphate isomerase, is expected to differentially stabilize the proposed stable reaction species: ketone, aldehyde, and enediol(ate). The identity and steady-state populations of the chemical entities bound to triosephosphate isomerase have been probed by using solid- and solution-state NMR. The 13C-enriched ketone substrate, dihydroxyacetone phosphate, was bound to the enzyme and characterized at steady state over a range of sample conditions. The ketone substrate was observed to be the major species over a temperature range from −60°C to 15°C. Thus, there is no suggestion that the enzyme preferentially stabilizes the reactive intermediate or the product. The predominance of dihydroxyacetone phosphate on the enzyme would support a mechanism in which the initial proton abstraction in the reaction from dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate is significantly slower than the subsequent chemical steps.

Rozovsky, S., Kaizuka, Y. & Groves, J. T. Formation and spatio-temporal evolution of periodic structures in lipid bilayers.  Journal of the American Chemical Society 127 (1), 36-37 (2005).

Abstract

Miscibility phase separation in lipid bilayers is widely implicated as an organizing principle in living cell membranes. However, the chemical and physical aspects of how membrane phase separation modulates protein activity remain obscure. Herein, we describe formation of ordered superstructures of coexisting liquid phases in bilayer membranes. Metastable stripe and hexagonal domain lattices are observed, as well as transitions between them. The high degree of order achieved by these methods facilitates statistical analysis of domain spatial distributions and enables measurement of domain interactions. Such long-range ordering principles may exist in more complicated membrane systems.

Desamero, R, Rozovsky, S., Zhadin, N., McDermott, A. E. & Callender, R. Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation.  Biochemistry 42 (10), 2941-2951 (2003).

Abstract

As for many enzymes, the enzymatic pathway of triosephosphate isomerase (TIM) includes the partially rate determining motion of an active site loop (loop 6, residues 166−176), which must remain closed during chemistry but must open just before product release. The motion of this loop was monitored using laser induced temperature-jump relaxation spectroscopy at nanosecond to millisecond time resolution. Trp168 in the hinge of the mobile loop served as a fluorophore reporter in a mutant of the yeast enzyme. The opening rate was studied as a function of the concentration of glycerol 3-phosphate, a substrate surrogate. Monoexponential kinetics were observed; assuming a simple two-step ligand release mechanism involving an encounter complex intermediate, the time scales of loop opening and closing were derived. The opening rate of the loop at 25 °C was determined to be 2500 ± 1000 s1, in remarkable agreement with solution and solid state NMR measurements. The closing rate at the same temperature was 46,700 ± 1800 s1. The rates were also studied as a function of the sample temperature following the jump. Enthalpies of activation of the loop motion, ΔHclose and ΔHopen, were estimated to be 13.8 and 14.1 kcal/mol, respectively. The enthalpy of dissociation estimated from the kinetic studies is in reasonable agreement with steady-state values. Moreover, the enthalpy was dissected, for the first time, into components associated with ion binding and with protein conformational change. The enthalpy of the release reaction appeared to have a substantial contribution from the dissociation of the ligand from the encounter complex, found to be endothermic at 6 kcal/mol. In contrast, the population ratio of the open to closed loop conformations is found to favor the closed conformation but to be substantially less temperature dependent than the release step. Preliminary data of other ligands show that G3P behavior resembles that of the substrate but differs from 2-phosphoglycolate, a tight binding inhibitor, and phosphate. This study represents one of the first detailed comparisons between NMR and fluorescence based probes of protein motion and results in good agreement between the methods. The data in aggregate support a model in which the rate of the loop opening for TIM is dependent on the ligand and results in opening rates in the presence of the product that are comparable to enzymatic throughput, kcat.

Jogl, G., Rozovsky, S., McDermott, A. E. & Tong, L. Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution.  Proceedings of the National Academy of Sciences 100, 50-55 (2003). 

Abstract

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the α-hydroxy carbons of the substrate (C… O ≈ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate’s O1 and O2 (N… O ≤ 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoidconfiguration. The ɛ ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate’s ketone oxygen, the bridging oxygen, and a terminal phosphate’s oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.

Rozovsky, S., Jogl, G., Tong, L. & McDermott, A. E. Solution-state NMR investigations of triosephosphate isomerase active site motion: ligand release in relation to active site loop dynamics.  Journal of Molecular Biology 310, 271-280 (2001).

Abstract

Product release is partially rate determining in the isomerization reaction catalyzed by Triosephosphate Isomerase, the conversion of dihydroxyacetone phosphate to d-glyceraldehyde 3-phosphate, probably because an active-site loop movement is necessary to free the product from confinement in the active-site. The timescale of the catalytic loop motion and of ligand release were studied using 19F and 31P solution-state NMR. A 5′-fluorotryptophan was incorporated in the loop N-terminal hinge as a reporter of loop motion timescale. Crystallographic studies confirmed that the structure of the fluorinated enzyme is indistinguishable from the wild-type; the fluorine accepts a hydrogen bond from water and not from a protein residue, with minimal perturbation to the flexible loop stability. Two distinct loop conformations were observed by 19F NMR. Both for unligated (empty) and ligated enzyme samples a single species was detected, but the chemical shifts of these two distinct species differed by 1.2 ppm. For samples in the presence of subsaturating amounts of a substrate analogue, glycerol 3-phosphate, both NMR peaks were present, with broadened lineshapes at 0°C. In contrast, a single NMR peak representing a rapid average of the two species was observed at 30°C. We conclude that the rate of loop motion is less than 1400 s−1 at 0°C and more than 1400 s−1 at 30°C. Ligand release was studied under similar sample conditions, using 31P NMR of the phosphate group of the substrate analogue. The rate of ligand release is less than 1000 s−1 at 0°C and more than 1000 s−1 at 30°C. Therefore, loop motion and product release are probably concerted and likely to represent a rate limiting step for chemistry.

Rozovsky, S. & McDermott, A. E. The timescale of a catalytic loop motion in triosephosphate isomerase.  Journal of Molecular Biology 310, 259-270 (2001).

Abstract

Loop 6 in the active site of Triosephosphate Isomerase (Saccharomyces cerevisiae) moves in order to reposition key residues for catalysis. The timescale of the opening and closing of this loop has been measured, at temperatures from −15 to +45°C, using broadline solid state deuterium NMR of a single deuterated tryptophan located in the loop’s N terminal hinge. The rate of the loop opening and closing was best detected using samples containing subsaturating amounts of a substrate analogue dl-glycerol 3-phosphate so that the populations of the open and closed forms were roughly equal, and using temperatures optimal for enzymatic function (30–45°C). The T2 values were much shorter than for unligated samples, consistent with full opening and closing of the loop at a rate of order 104 s−1, and in good agreement with the rates estimated based on solution state 19F NMR. The loop motion appears to be partially rate limiting for chemistry in both directions.

Roni Aloni, Pua Feigenbaum, Natalie Kalev and Sharon Rozovsky. Hormonal control of vascular differentiation in plants: the physiological basis of cambium ontogeny and xylem evolution. In “Cell and Molecular Biology of Wood Formation” Editors Savidge, R. A., Barnett, J. R. & Napier, R. BIOS Scientific Publishers, Oxford, pp 223-236 (2000)