Publications by authors named "Donald E Spratt"

32 Publications

Structural Insights into Ankyrin Repeat-Containing Proteins and Their Influence in Ubiquitylation.

Int J Mol Sci 2021 Jan 9;22(2). Epub 2021 Jan 9.

Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610, USA.

Ankyrin repeat (AR) domains are considered the most abundant repeat motif found in eukaryotic proteins. AR domains are predominantly known to mediate specific protein-protein interactions (PPIs) without necessarily recognizing specific primary sequences, nor requiring strict conformity within its own primary sequence. This promiscuity allows for one AR domain to recognize and bind to a variety of intracellular substrates, suggesting that AR-containing proteins may be involved in a wide array of functions. Many AR-containing proteins serve a critical role in biological processes including the ubiquitylation signaling pathway (USP). There is also strong evidence that AR-containing protein malfunction are associated with several neurological diseases and disorders. In this review, the structure and mechanism of key AR-containing proteins are discussed to suggest and/or identify how each protein utilizes their AR domains to support ubiquitylation and the cascading pathways that follow upon substrate modification.
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http://dx.doi.org/10.3390/ijms22020609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826745PMC
January 2021

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.

PLoS One 2020 8;15(7):e0235925. Epub 2020 Jul 8.

Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, Worcester, MA, United States of America.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343168PMC
September 2020

HECT E3 ubiquitin ligases - emerging insights into their biological roles and disease relevance.

J Cell Sci 2020 04 7;133(7). Epub 2020 Apr 7.

Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610, USA

Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases play a critical role in various cellular pathways, including but not limited to protein trafficking, subcellular localization, innate immune response, viral infections, DNA damage responses and apoptosis. To date, 28 HECT E3 ubiquitin ligases have been identified in humans, and recent studies have begun to reveal how these enzymes control various cellular pathways by catalyzing the post-translational attachment of ubiquitin to their respective substrates. New studies have identified substrates and/or interactors with different members of the HECT E3 ubiquitin ligase family, particularly for E6AP and members of the neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) family. However, there still remains many unanswered questions about the specific roles that each of the HECT E3 ubiquitin ligases have in maintaining cellular homeostasis. The present Review discusses our current understanding on the biological roles of the HECT E3 ubiquitin ligases in the cell and how they contribute to disease development. Expanded investigations on the molecular basis for how and why the HECT E3 ubiquitin ligases recognize and regulate their intracellular substrates will help to clarify the biochemical mechanisms employed by these important enzymes in ubiquitin biology.
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http://dx.doi.org/10.1242/jcs.228072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157599PMC
April 2020

Crystal structure of the catalytic C-lobe of the HECT-type ubiquitin ligase E6AP.

Protein Sci 2020 06 5;29(6):1550-1554. Epub 2020 Feb 5.

Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.

The HECT-type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus-induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C-terminal lobe ("C-lobe") of the catalytic domain of E6AP that reveals two molecules in a domain-swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active-site region. While such dimerization is unlikely to occur in the context of full-length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C-lobe of another HECT-type ubiquitin ligase, HERC6. This may point to conformational strain in the active-site region of HECT-type ligases with possible implications for catalysis. SIGNIFICANCE STATEMENT: The HECT-type ubiquitin ligase E6AP has key roles in human papilloma virus-induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C-terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT-type ligases.
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http://dx.doi.org/10.1002/pro.3832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255509PMC
June 2020

Rapid and efficient purification of Drosophila homeodomain transcription factors for biophysical characterization.

Protein Expr Purif 2019 06 7;158:9-14. Epub 2019 Feb 7.

Gustaf H. Carlson School of Chemistry & Biochemistry, Clark University, 950 Main St, Worcester, MA, 01610, USA. Electronic address:

Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through their interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6 M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult. To circumvent these obstacles, we have developed a streamlined expression and purification protocol to produce large yields of Drosophila HD TFs. Using the HD TFs FUSHI-TARAZU (FTZ), ANTENNAPEDIA (ANTP), ABDOMINAL-A (ABD-A), ABDOMINAL-B (ABD-B), and ULTRABITHORAX (UBX) as examples, we demonstrate that our 3-day protocol involving the overexpression of His-SUMO fusion constructs in E. coli followed by a Ni-IMAC, SUMO-tag cleavage with the SUMO protease Ulp1, and a heparin column purification produces pure, soluble protein in biological buffers around pH 7 in the absence of denaturants. Electrophoretic mobility shift assays (EMSA) confirm that the purified HD proteins are functional and nuclear magnetic resonance (NMR) spectra confirm that the purified HDs are well-folded. These purified HD TFs can be used in future biophysical experiments to structurally and biochemically characterize how and why these HD TFs bind to different DNA sequences and further probe how nucleotide differences contribute to TF-DNA specificity in the HD family.
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http://dx.doi.org/10.1016/j.pep.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424608PMC
June 2019

A subset of calcium-binding S100 proteins show preferential heterodimerization.

FEBS J 2019 05 21;286(10):1859-1876. Epub 2019 Feb 21.

Department of Biochemistry, The University of Western Ontario, London, Canada.

The assembly of proteins into dimers and oligomers is a necessary step for the proper function of transcription factors, muscle proteins, and proteases. In uncontrolled states, oligomerization can also contribute to illnesses such as Alzheimer's disease. The S100 protein family is a group of dimeric proteins that have important roles in enzyme regulation, cell membrane repair, and cell growth. Most S100 proteins have been examined in their homodimeric state, yet some of these important proteins are found in similar tissues implying that heterodimeric molecules can also be formed from the combination of two different S100 members. In this work, we have established co-expression methods in order to identify and quantify the distribution of homo- and heterodimers for four specific pairs of S100 proteins in their calcium-free states. The split GFP trap methodology was used in combination with other GFP variants to simultaneously quantify homo- and heterodimeric S100 proteins in vitro and in living cells. For the specific S100 proteins examined, NMR, mass spectrometry, and GFP trap experiments consistently show that S100A1:S100B, S100A1:S100P, and S100A11:S100B heterodimers are the predominant species formed compared to their corresponding homodimers. We expect the tools developed here will help establish the roles of S100 heterodimeric proteins and identify how heterodimerization might alter the specificity for S100 protein action in cells.
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http://dx.doi.org/10.1111/febs.14775DOI Listing
May 2019

Resveratrol Sustains Insulin-Degrading Enzyme Activity toward Aβ42.

ACS Omega 2018 Oct 16;3(10):13275-13282. Epub 2018 Oct 16.

Carlson School of Chemistry and Biochemistry, Clark University, 950 Main Street, Worcester, Massachusetts 01610, United States.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is the sixth leading cause of death in the United States. We hypothesize that the impaired clearance of Aβ42 from the brain is partly responsible for the onset of sporadic AD. In this work, we evaluated the activity of insulin-degrading enzyme (IDE) toward Aβ42 in the presence of resveratrol, a polyphenol found in red wine and grape juice. By liquid chromatography/mass spectrometry, we identified initial cleavage sites in the absence and presence of resveratrol that carry biological relevance connected to the amyloidogenic properties of Aβ42. Incubation with resveratrol results in a substantial increase in Aβ42 fragmentation compared to the control, signifying that the polyphenol sustains IDE-dependent degradation of Aβ42 and its fragments. Our findings suggest that therapeutic and/or preventative approaches combining resveratrol and IDE may hold promise for sporadic AD.
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http://dx.doi.org/10.1021/acsomega.8b01913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210067PMC
October 2018

Enzyme kinetics from circular dichroism of insulin reveals mechanistic insights into the regulation of insulin-degrading enzyme.

Biosci Rep 2018 12 7;38(6). Epub 2018 Nov 7.

Carlson School of Chemistry and Biochemistry, Clark University, 950 Main Street, Worcester, MA 01610, U.S.A.

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that selectively degrades biologically important substrates associated with type 2 diabetes and Alzheimer's disease (AD). As such, IDE is an attractive target for therapeutic innovations. A major requirement is an understanding of how other molecules present in cells regulate the activity of the enzyme toward insulin, IDE's most important physiologically relevant substrate. Previous kinetic studies of the IDE-dependent degradation of insulin in the presence of potential regulators have used iodinated insulin, a chemical modification that has been shown to alter the biological and biochemical properties of insulin. Here, we present a novel kinetic assay that takes advantage of the loss of helical circular dichroic signals of insulin with IDE-dependent degradation. As proof of concept, the resulting Michaelis-Menten kinetic constants accurately predict the known regulation of IDE by adenosine triphosphate (ATP). Intriguingly, we found that when Mg is present with ATP, the regulation is abolished. The implication of this result for the development of preventative and therapeutic strategies for AD is discussed. We anticipate that the new assay presented here will lead to the identification of other small molecules that regulate the activity of IDE toward insulin.
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http://dx.doi.org/10.1042/BSR20181416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239264PMC
December 2018

H, C, and N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH.

Biomol NMR Assign 2019 04 18;13(1):15-20. Epub 2018 Sep 18.

Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA, 01610, USA.

ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family that regulates numerous cellular functions including inflammatory responses through T-cell activation, cell differentiation, and apoptosis. Known intracellular targets of ITCH-dependent ubiquitylation include receptor proteins, signaling molecules, and transcription factors. The HECT C-terminal lobe of ITCH contains the conserved catalytic cysteine required for the covalent attachment of ubiquitin onto a substrate and polyubiquitin chain assembly. We report here the complete experimentally determined H, C, and N backbone and sidechain resonance assignments for the HECT C-terminal lobe of ITCH (residues 784-903) using heteronuclear, multidimensional NMR spectroscopy. These resonance assignments will be used in future NMR-based studies to examine the role of dynamics and conformational flexibility in HECT-dependent ubiquitylation as well as deciphering the structural and biochemical basis for polyubiquitin chain synthesis and specificity by ITCH.
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http://dx.doi.org/10.1007/s12104-018-9843-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439258PMC
April 2019

The Longest Amyloid-β Precursor Protein Intracellular Domain Produced with Aβ42 Forms β-Sheet-Containing Monomers That Self-Assemble and Are Proteolyzed by Insulin-Degrading Enzyme.

ACS Chem Neurosci 2018 12 3;9(12):2892-2897. Epub 2018 Aug 3.

Carlson School of Chemistry and Biochemistry , Clark University , 950 Main Street , Worcester , Massachusetts 01610 , United States.

Alzheimer's disease (AD) is the most common neurodegenerative disease resulting in dementia. It is characterized pathologically by extracellular amyloid plaques composed mainly of deposited Aβ42 and intracellular neurofibrillary tangles formed by hyperphosphorylated tau protein. Recent clinical trials targeting Aβ have failed, suggesting that other polypeptides produced from the amyloid-β precursor protein (APP) may be involved in AD. An attractive polypeptide is AICD57, the longest APP intracellular domain (AICD) coproduced with Aβ42. Here, we show that AICD57 forms micelle-like assemblies that are proteolyzed by insulin-degrading enzyme (IDE), indicating that AICD57 monomers are in dynamic equilibrium with AICD57 assemblies. The N-terminal part of AICD57 monomer is not degraded, but its C-terminal part is hydrolyzed, particularly in the YENPTY motif that has been associated with the hyperphosphorylation of tau. Therefore, sustaining IDE activity well into old age holds promise for regulating levels of not only Aβ but also AICD in the aging brain.
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http://dx.doi.org/10.1021/acschemneuro.8b00305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7074877PMC
December 2018

Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping.

FEBS Lett 2016 05 4;590(10):1530-42. Epub 2016 May 4.

Department of Biochemistry, The University of Western Ontario, London, Canada.

The activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb(S65) ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (ΔRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb(S20) failed to stimulate parkin, parkin was partially active with pUb(S12) . We observed significant ubiquitination when pUb(S65) was the sole substrate.
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http://dx.doi.org/10.1002/1873-3468.12182DOI Listing
May 2016

Suramin inhibits cullin-RING E3 ubiquitin ligases.

Proc Natl Acad Sci U S A 2016 Apr 21;113(14):E2011-8. Epub 2016 Mar 21.

Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY 10029; Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical College, Xuzhou, 221002, China;

Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.
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http://dx.doi.org/10.1073/pnas.1601089113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833235PMC
April 2016

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

EMBO J 2015 Oct 7;34(20):2506-21. Epub 2015 Aug 7.

MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences University of Dundee, Dundee, UK

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin-parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.
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http://dx.doi.org/10.15252/embj.201592337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609183PMC
October 2015

Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination.

Proc Natl Acad Sci U S A 2014 Jun 27;111(23):8434-9. Epub 2014 May 27.

Departments of Oncological Sciences,Xuzhou Medical College, Jiangsu Key Laboratory of Biological Cancer Therapy, Jiangsu 221002, China

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(βTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.
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http://dx.doi.org/10.1073/pnas.1407849111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060658PMC
June 2014

RBR E3 ubiquitin ligases: new structures, new insights, new questions.

Biochem J 2014 Mar;458(3):421-37

*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada, N6A 5C1.

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.
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http://dx.doi.org/10.1042/BJ20140006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940038PMC
March 2014

A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination.

J Biol Chem 2014 Mar 24;289(10):7068-81. Epub 2014 Jan 24.

From the Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, New York 10029-6574.

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF(βTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(βTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(βTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
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http://dx.doi.org/10.1074/jbc.M113.530576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945367PMC
March 2014

Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase.

PLoS One 2013 15;8(8):e74047. Epub 2013 Aug 15.

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074047PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772753PMC
June 2014

A molecular explanation for the recessive nature of parkin-linked Parkinson's disease.

Nat Commun 2013 ;4:1983

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.
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http://dx.doi.org/10.1038/ncomms2983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3709501PMC
December 2013

Structure and dynamics of calmodulin (CaM) bound to nitric oxide synthase peptides: effects of a phosphomimetic CaM mutation.

Biochemistry 2012 May 16;51(17):3651-61. Epub 2012 Apr 16.

Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes.
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http://dx.doi.org/10.1021/bi300327zDOI Listing
May 2012

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase.

J Biol Chem 2012 May 20;287(21):17374-17385. Epub 2012 Mar 20.

Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada. Electronic address:

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.
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http://dx.doi.org/10.1074/jbc.M112.353748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366790PMC
May 2012

Association of the disordered C-terminus of CDC34 with a catalytically bound ubiquitin.

J Mol Biol 2011 Apr 4;407(3):425-38. Epub 2011 Feb 4.

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Cell division cycle protein 34 (CDC34) is a key E2 ubiquitin (Ub)-conjugating enzyme responsible for the polyubiquitination of proteins controlling the G1/S stages of cell division. The acidic C-terminus of the enzyme is required for this function, although there is little structural information providing details for a mechanism. One logical time point involving the C-terminus is the CDC34-Ub thiolester complex that precedes Ub transfer to a substrate. To examine this, we used a CDC34-Ub disulfide complex that structurally mimics the thiolester intermediate. NMR spectroscopy was used to show that the CDC34 C-terminus is disordered but can intramolecularly interact with the catalytically bound Ub. Using chemical shift perturbation analysis, we mapped two interacting regions on the surface of Ub in the CDC34-Ub complex. The first site comprises a hydrophobic patch (typical of other Ub complexes) that associates with the CDC34 catalytic domain. A novel second site, dependent on the C-terminus of CDC34, comprises a lysine-rich surface (K6, K11, K29, and K33) on the opposite face of Ub. Further, NMR experiments show that this interaction is described by two slowly exchanging states-a compact conformation where the C-terminus of CDC34 interacts with bound Ub and an extended structure where the C-terminus is released. This work provides the first structural details that show how the C-terminus of CDC34 might direct a thiolester-bound Ub to control polyubiquitin chain formation.
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http://dx.doi.org/10.1016/j.jmb.2011.01.047DOI Listing
April 2011

Codon optimization for enhanced Escherichia coli expression of human S100A11 and S100A1 proteins.

Protein Expr Purif 2010 Sep 27;73(1):58-64. Epub 2010 Mar 27.

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

The cloning, expression and purification for the recombinant full-length human proteins S100A11 and human S100A1 is described. The genes were synthesized by overlapping complementary single-stranded oligonucleotides of various lengths. The coding sequence for both genes were codon optimized by selecting only the most preferential codons according to the Escherichia coli bias. In order to assemble the various oligonucleotides into the correct full-length genes, a unique one-step PCR procedure was implemented. The expression and purification procedures were also optimized for each protein. A single phenyl-Sepharose column was sufficient for the purification of human S100A11 whereas HiTrap Q anion exchange followed by phenyl-Sepharose columns were required for the purification of S100A1. By optimizing the S100A1 and S100A11 gene, expression and purification protocols, more than 45 and 150mg, respectively of the purified human proteins were obtained per litre of media. Protein identity was verified by both SDS-PAGE and mass spectrometry (MS) and further characterized by NMR spectroscopy. These results have established an efficient method for the expression and purification of large quantities of human S100A1 and S100A11 proteins for biophysical characterization.
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http://dx.doi.org/10.1016/j.pep.2010.03.015DOI Listing
September 2010

FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes.

Biochemistry 2008 Nov 24;47(46):12006-17. Epub 2008 Oct 24.

Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.
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http://dx.doi.org/10.1021/bi801418sDOI Listing
November 2008

Regulation of mammalian nitric oxide synthases by electrostatic interactions in the linker region of calmodulin.

Biochim Biophys Acta 2008 Dec 19;1784(12):2065-70. Epub 2008 Sep 19.

Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.

Calmodulin (CaM), the ubiquitous Ca(2+)-sensing protein, consists of two globular domains separated by a flexible central linker that properly orients CaM's globular domains to bind and regulate various intracellular proteins, including the nitric oxide synthase (NOS) enzymes. In the present study we determined that the charge and length of the central linker of CaM has an effect on the binding and activation of the NOS isozymes by using a variety of charge CaM mutants (T79D, S81D, T79D/S81D, S101D and E84R/E87K) and CaM mutants with residues removed (Delta84, Delta83-84, and Delta81-84). Our kinetic and spectropolarimetry results demonstrate that the NOS enzymes are not adversely affected by the CaM mutants with the exceptions of S101D, E84R/E87K and the deletion of residue 84. Electrostatic interactions in the central linker between residues 82-87 in combination with hydrophobic interactions in the globular domains of CaM are important for its tight association to inducible NOS.
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http://dx.doi.org/10.1016/j.bbapap.2008.09.002DOI Listing
December 2008

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme.

J Biol Inorg Chem 2009 Jan 2;14(1):133-42. Epub 2008 Oct 2.

College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808-3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 +/- 5 and 35 +/- 3 s(-1), respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.
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http://dx.doi.org/10.1007/s00775-008-0431-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596912PMC
January 2009

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases.

Biochim Biophys Acta 2007 Oct 19;1774(10):1351-8. Epub 2007 Aug 19.

Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.
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http://dx.doi.org/10.1016/j.bbapap.2007.07.019DOI Listing
October 2007

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes.

Biochemistry 2007 Jul 20;46(28):8288-300. Epub 2007 Jun 20.

Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.
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http://dx.doi.org/10.1021/bi062130bDOI Listing
July 2007

Selective labeling of selenomethionine residues in proteins with a fluorescent derivative of benzyl bromide.

Anal Biochem 2006 Dec 18;359(2):253-8. Epub 2006 Oct 18.

Department of Chemistry, University of Waterloo, Waterloo, Ont., Canada N2L 3G1.

Benzyl bromide is used as a reagent for the selective modification of methionine residues in proteins. We here explored the suitability of the bromobenzyl moiety as a reactive group for the targeted fluorescent labeling of methionine and selenomethionine residues in proteins. A novel labeling reagent (N,N',N'-trimethyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- N'-(p-bromomethylbenzyl)-ethylenediamine, NBD-BBr) was synthesized and tested for reactivity with two model proteins containing single methionine or selenomethionine residues. The amounts of reagent and reactions times required for modification of methionine resulted in side reactions with other amino acid residues, a finding which was also confirmed for benzyl bromide itself. However, with selenomethionine, lower concentrations and shorter reaction times were sufficient for NBD-BBr modification. Under these conditions, labeling was confined to selenomethionine residues with one but not the other model protein. Where applicable, the protein labeling strategy characterized here is rapid and efficient. It should be useful in combination with cysteine-specific labeling if dual site-specific modification is desired.
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http://dx.doi.org/10.1016/j.ab.2006.09.025DOI Listing
December 2006

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs.

FEBS J 2006 Apr;273(8):1759-71

Department of Chemistry, University of Waterloo, ON, Canada.

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05193.xDOI Listing
April 2006