Interaction Of N-terminal Domain Research Articles

Abstract LB_C22: Dynamic interactions of disordered N-terminal domain of p53 in cancer

Abstract TP53 is mutated in over half of all human cancers, making it the most important tumor suppressor. p53 sequence includes an N-terminal domain (NTD), a DNA binding domain (DBD), and others. NTD is intrinsically disordered and interacts with many proteins, acting as a hub for cellular signaling. Using a combination of computational and experimental approaches, we show NTD have dynamic interactions with cyclophilin D (CypD), a mitochondria proline isomerase with key roles in cellular necrosis and mitochondrial energy metabolism. We show that a subset of p53 truncation mutations can promote cancer cell proliferation and mitochondrial metabolism in a CypD-dependent manner. Although dynamic interfaces are often considered “undruggable”, we have demonstrated that EGCG, the active ingredient of green tea, can bind to NTD and disrupt NTD-CypD interaction, inhibiting the effects of p53 truncation mutants. Our studies point to a novel mechanism for p53 gain-of-function (GOF) mutations through NTD interaction with mitochondrial CypD. Our data also provide novel insights into dynamic interactions of intrinscially disordered proteins and small molecule engagement of such challenging drug targets, with important implications for cancer treatment. Citation Format: Chunyu Wang, Baines Christopher, Jianhan Chen, Stewart Loh. Dynamic interactions of disordered N-terminal domain of p53 in cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C22.

The cytoplasmic N-terminal tail of Zika virus NS4A protein forms oligomers in the absence of detergent or lipids

The non-structural (NS) NS4A protein in flaviviruses has three predicted transmembrane domains, is critical for virulence and participates in membrane morphogenesis. In Dengue virus (DENV), both hydrophylic N-terminal tail and its first transmembrane domain participate in the formation of oligomers which are important for pathogenicity. However, the relative importance of the N-terminal domain in oligomerization has been under debate. In particular, since in the absence of detergent or lipids, this domain (residues 1–48) in both DENV and Zika virus (ZIKV) NS4A, was found to be disordered. Recently, however, we reported preliminary data that showed that peptide ZIKV NS4A 4–58 adopts a defined secondary structure in aqueous solution and forms oligomers, signaling its importance for full length NS4A oligomerization. Herein we have performed detailed analytical ultracentrifugation experiments to further characterize the oligomerization of this peptide and also a shorter variant (residues 4–44). In both cases, sedimentation velocity produced a single species with concentration-dependent sedimentation coefficient, consistent with a fast equilibrium between at least two species. Combining sedimentation velocity and equilibrium experiments, data is best fitted to a monomer–dimer–trimer equilibrium. Possible models of NS4A oligomers obtained with AlphaFold-2 predict the stabilizing role for residues in this N-terminal domain, such as Arg20, Asn27, Ala44 and Glu50, all at highly conserved positions in flavivirus NS4A proteins. Our results are thus consistent with N-terminal domain interactions acting as one of the driving forces for NS4A homo-oligomerization.

Myosin binding protein h dominates the "c-zone" in ultrafast swimming muscles of developing zebrafish.

The ‘C-zone’ of mature vertebrate skeletal muscle sarcomeres is characterized by the presence of Myosin-Binding Protein C (MyBP-C), which ‘tunes’ contractility via its N-terminal domain interactions with the myosin head region and/or the thin filament. The ultrafast swimming muscles of zebrafish larvae are a perfect model system to define MyBP-C isoform function, due to tractable genetics and easily characterized muscle mechanics. However, liquid chromatography mass spectrometry shows 5-day-old larval muscle expresses MyBP-C at only ∼5% of the ∼1:10 molar ratio with myosin heavy chain (MYH) typically associated with full C-zone occupancy, while a little-studied MyBP-C paralog, Myosin-Binding Protein H (MyBP-H) comprises the remaining ∼95%. MyBP-H is homologous to MyBP-C's C terminus, which binds to the myosin thick filament backbone, but it lacks analagous N-terminal regulatory domains, which are critical to MyBP-C's slowing of muscle shortening. Therefore, the presence of MyBP-H may underlie the extreme contractile speed of larval muscle (>30 lengths/s), and/or indicate its involvement in muscle development. To define MyBP-H's potential roles, we first imaged its sarcomeric localization in larvae expressing transgenic, FLAG-tagged MyBP-H. Immunofluorescence revealed characteristic fluorescence doublets at the sarcomere center, indicating MyBP-H binds throughout the C-zone. Interestingly, MyBP-H-null mutants engineered using CRISPR/Cas9 appear to develop normally, suggesting the developmental impact of MyBP-H may be subtle. Preliminary mechanical studies of MyBP-H-null larval muscles showed no change in twitch kinetics, tetanic force, or the Force:Velocity relationship, which might be expected, given MyBP-C content increases only minimally (From ∼5% to ∼10% of full C-zone occupancy). Although the role of MyBP-H in development remains an enigma, physiologically stressing the larvae may reveal MyBP-H's function. At a minimum, the MyBP-H null larvae provide a clean slate to repopulate the C-zone using transgenically expressed human or disease-causing MyBP-C isoforms.

The Magnetosome Protein, Mms6 from Magnetospirillum magneticum Strain AMB-1, Is a Lipid-Activated Ferric Reductase.

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.

AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP \u03b38 interactions

AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.

N-terminal Domain of TDP43 Enhances Liquid-Liquid Phase Separation of Globular Proteins

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43′s NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.

Podocyte RNF166 deficiency alleviates diabetic nephropathy by mitigating mitochondria impairment and apoptosis via regulation of CYLD signal

Diabetic nephropathy (DN) is a major cause of renal failure in diabetic patients. RING-finger protein 166 (RNF166), composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif, plays a critical role in mediating various cellular processes. However, its potential in DN has not been investigated. In the present study, we found that DN patients exhibited significantly increased expression of RNF166 in renal tissues compared with the normal individuals, and abundant RNF166 was detected in podocytes. We then showed that podocyte-conditional RNF166 knockout (RNF166cKO) markedly reduced blood glucose levels and ameliorated renal dysfunction in streptozotocin (STZ)-induced diabetic mice. Additionally, abnormal histological changes and podocyte injury were observed in STZ-induced diabetic mice, while being markedly ameliorated by RNF166cKO. Furthermore, podocyte-specific RNF166 deficiency considerably mitigated apoptosis and mitochondrial impairments in glomeruli podocytes of STZ-challenged mice through suppressing Caspase-3 cleavage and improving mitochondrial fission-associated molecules. In vitro studies further confirmed that high glucose (HG) induced mitochondrial dysfunction, along with enhanced releases of Cyto-c from mitochondria and elevated expression of cleaved Caspase-9, contributing to intrinsic apoptosis in podocytes. Intriguingly, these effects triggered by HG were dramatically ameliorated by RNF166 knockout. Mechanistically, we demonstrated that RNF166 directly interacted with cylindromatosis (CYLD), and negatively regulated CYLD expression. Notably, RNF166 knockout-attenuated mitochondrial damage and apoptosis were mainly through CYLD in podocytes upon HG stimulation. Together, all these findings provided new insights into the novel effects of RNF166 on maintaining mitochondrial function and apoptosis in podocytes during DN progression both in vivo and in vitro through interacting with CYLD, indicating that RNF166/CYLD may be an innovative therapeutic target for developing effective strategy against DN development.

RING-finger protein 166 plays a novel pro-apoptotic role in neurotoxin-induced neurodegeneration via ubiquitination of XIAP

The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been widely utilized to establish experimental models of Parkinson disease and to reveal the critical molecules and pathway underlying neuronal death. The profile of gene expression changes following 6-OHDA treatment of MN9D dopaminergic neuronal cells was investigated using a TwinChip Mouse-7.4K microarray. Functional clustering of altered sets of genes identified RING-finger protein 166 (RNF166). RNF166 is composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif. RNF166 localized in the cytosol and nucleus. At the tissue level, RNF166 was widely expressed in the central nervous system and peripheral organs. In the cerebral cortex, its expression decreased over time. In certain conditions, overexpression of RNF166 accelerates the naturally occurring neuronal death and 6-OHDA–induced MN9D cell death as determined by TUNEL and annexin-V staining, and caspase activation. Consequently, 6-OHDA–induced apoptotic cell death was attenuated in RNF166-knockdown cells. In an attempt to elucidate the mechanism underlying this pro-apoptotic activity, binding protein profiles were assessed using the yeast two-hybrid system. Among several potential binding candidates, RNF166 was shown to interact with the cytoplasmic X-linked inhibitor of apoptosis (XIAP), inducing ubiquitin-dependent degradation of XIAP and eventually accelerating caspase activation following 6-OHDA treatment. RNF166’s interaction with and resulting inhibition of the XIAP anti-caspase activity was further enhanced by XIAP-associated factor-1 (XAF-1). Consequently, depletion of RNF166 suppressed 6-OHDA–induced caspase activation and apoptotic cell death, which was reversed by XIAP knockdown. In summary, our data suggest that RNF166, a novel E3 ligase, plays a pro-apoptotic role via caspase activation in neuronal cells.

Class IA PI3K regulatory subunits: p110-independent roles and structures.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.

Tyrosyl-DNA Phosphodiesterase I N-Terminal Domain Modifications and Interactions Regulate Cellular Function.

The conserved eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1) removes a diverse array of adducts from the end of DNA strand breaks. Tdp1 specifically catalyzes the hydrolysis of phosphodiester linked DNA-adducts. These DNA lesions range from damaged nucleotides to peptide-DNA adducts to protein-DNA covalent complexes and are products of endogenously or exogenously induced insults or simply failed reaction products. These adducts include DNA inserted ribonucleotides and non-conventional nucleotides, as well as covalent reaction intermediates of DNA topoisomerases with DNA and a Tdp1-DNA adduct in trans. This implies that Tdp1 plays a role in maintaining genome stability and cellular homeostasis. Dysregulation of Tdp1 protein levels or catalysis shifts the equilibrium to genome instability and is associated with driving human pathologies such as cancer and neurodegeneration. In this review, we highlight the function of the N-terminal domain of Tdp1. This domain is understudied, structurally unresolved, and the least conserved in amino acid sequence and length compared to the rest of the enzyme. However, over time it emerged that the N-terminal domain was post-translationally modified by, among others, phosphorylation, SUMOylation, and Ubiquitinoylation, which regulate Tdp1 protein interactions with other DNA repair associated proteins, cellular localization, and Tdp1 protein stability.

Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705-SH2 through C-terminal tail modulation.

Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705-SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT-CTT intermolecular and the CTT-SH2 intramolecular interactions that support pY705-SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705-SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705-SH2 interaction. Thus, pS727 triggers pY705-SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation-inactivation for proper function of STAT3.

Impact of deamidation on gliadin-based nanoparticle formation and curcumin encapsulation

The physicochemical properties of gliadin-based nanoparticles and mechanisms involved in the particle formation and curcumin encapsulation were investigated. The mean particle sizes and ζ-potential indicated that stable nanoparticles can be produced around pH 7–9 where native gliadin particles were unstable. The Fourier-transform infrared spectroscopy showed that deamidation can alter particle formation with more intraparticle interactions of N-terminal domain in deamidated gliadins (DG). Results revealed that hydrophobic interaction and hydrogen bond were the major driving forces for the particle formation in gliadins. The curcumin-to-protein mass ratio of 1/20 is the optimal ratio for gliadins to encapsulate curcumin with over 91% encapsulation efficiency. Curcumin loaded in DG nanoparticles showed higher thermal stability compared with native ones. Thermodynamic parameters suggested curcumin can bind with gliadin, DG with degrees of deamidation (DD) of 8.8% and 10.7% mainly through hydrogen bonding while for DG with DD of 5.7%, the main driven force was hydrophobic interaction.

NLRP10 Affects the Stability of Abin-1 To Control Inflammatory Responses.

NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-κB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-κB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses.

The structural basis of human Spt16 N-terminal domain interaction with histone (H3-H4)2 tetramer

FACT (Facilitates Chromatin Transactions) is a heterodimeric protein complex involved in RNA polymerase II transcription elongation, playing essential roles in chromatin remodeling during transcription, replication, and DNA damage repair. The FACT subunit hSpt16 is essential for nucleosome reorganization. The N-terminal domain of hSpt16 (hSpt16-NTD) was recently described as a histone (H3-H4)2-binding domain; however, its mode of interaction remains unknown. In this study, we solved the structure of hSpt16-NTD437 at 2.19 Å and found that a long-disordered region (hSpt16-LDR), after the main body of hSpt16-NTD, is a novel histone-binding motif. Furthermore, hSpt16-LDR interaction with (H3-H4)2 is H3 N-terminal tail-independent. Therefore, Spt16-NTD is a histone H3-H4-specific binding domain with a distinct mechanism of interaction between histones and histone chaperones.

Coupling chemosensory array formation and localization.

Chemotaxis proteins organize into large, highly ordered, chemotactic signaling arrays, which in Vibrio species are found at the cell pole. Proper localization of signaling arrays is mediated by ParP, which tethers arrays to a cell pole anchor, ParC. Here we show that ParP's C-terminus integrates into the core-unit of signaling arrays through interactions with MCP-proteins and CheA. Its intercalation within core-units stimulates array formation, whereas its N-terminal interaction domain enables polar recruitment of arrays and facilitates its own polar localization. Linkage of these domains within ParP couples array formation and localization and results in controlled array positioning at the cell pole. Notably, ParP's integration into arrays modifies its own and ParC's subcellular localization dynamics, promoting their polar retention. ParP serves as a critical nexus that regulates the localization dynamics of its network constituents and drives the localized assembly and stability of the chemotactic machinery, resulting in proper cell pole development.

Identification of a novel small-molecule compound targeting the influenza A virus polymerase PB1-PB2 interface

The PB1 C-terminal domain and PB2 N-terminal domain interaction of the influenza A polymerase, which modulates the assembly of PB1 and PB2 subunits, may serve as a valuable target for the development of novel anti-influenza therapeutics. In this study, we performed a systematic screening of a chemical library, followed by the antiviral evaluation of primary hits and their analogues. Eventually, a novel small-molecule compound PP7 that abrogated the PB1-PB2 association and impaired viral polymerase activity was identified. PP7 exhibited antiviral activities against influenza virus subtypes A (H1N1)pdm09, A(H7N9) and A(H9N2) in cell cultures and partially protected mice against lethal challenge of mouse-adapted influenza A (H1N1)pdm09 virus. Surprisingly, a panel of other subtypes of influenza virus, including A(H5N1) and A(H7N7), showed various degrees of resistance to the compound. Biochemical studies revealed a similar pattern of resistance on the impairment of polymerase activity. Molecular docking analyses suggested a PP7-binding site that appeared to be completely conserved among the subtypes of the virus mentioned above. Thus, we propose that alternative/additional binding site (s) may exist for the regulation of PB1-PB2 subunits assembly of influenza A virus.

A Hybrid Methods Approach to Determine the Structure of Tetrahymena Telomerase

Telomerase is an RNA-protein complex that extends the ends of linear chromosomes, and is a highly regulated determinant of cellular aging, stem cell renewal, and tumorigenesis. We are using a combination of NMR spectroscopy, X-ray crystallography, mass spectrometry, and electron microscopy to study the structure and function of telomerase. We previously reported the 3D structure of endogenously assembled Tetrahymena thermophila telomerase holoenzyme at 25 A resolution using negative stain electron microscopy (EM) (Jiang et al Nature 2013). Six of the 7 protein subunits and the stem-loop 2 region of telomerase RNA (TER) were localized in the 3D structure by affinity labeling. Among the holoenzyme proteins, p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution correlates activity with structure. This first physical and functional network architecture of a telomerase holoenzyme provided the first view into the structure of the telomerase reverse transcriptase (TERT)-TER-p65 catalytic core and revealed the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres. More recently, we determined the cryoelectron microscopy structure of Tetrahymena telomerase at ∼9-A resolution. In addition to the 7 known holoenzyme proteins, 2 new proteins are identified, which form a complex (TEB) with single-stranded telomere DNA-binding protein Teb1, paralogous to heterotrimeric Replication Protein A (RPA). The p75-p45-p19 subcomplex is identified as another RPA-related complex, CST. This study reveals the paths of TER in the TERT-TER-p65 catalytic core and ssDNA exit, extensive subunit interactions of TERT essential N-terminal domain, p50, and TEB, and new subunit identities and structures, including p19 and p45C crystal structures, providing unprecedented structural and mechanistic insights into telomerase holoenzyme function.

The cMyBP-C HCM variant L348P enhances thin filament activation through an increased shift in tropomyosin position

Mutations in cardiac myosin binding protein C (cMyBP-C), a thick filament protein that modulates contraction of the heart, are a leading cause of hypertrophic cardiomyopathy (HCM). Electron microscopy and 3D reconstruction of thin filaments decorated with cMyBP-C N-terminal fragments suggest that one mechanism of this modulation involves the interaction of cMyBP-C's N-terminal domains with thin filaments to enhance their Ca2+-sensitivity by displacement of tropomyosin from its blocked (low Ca2+) to its closed (high Ca2+) position. The extent of this tropomyosin shift is reduced when cMyBP-C N-terminal domains are phosphorylated. In the current study, we have examined L348P, a sequence variant of cMyBP-C first identified in a screen of patients with HCM. In L348P, leucine 348 is replaced by proline in cMyBP-C's regulatory M-domain, resulting in an increase in cMyBP-C's ability to enhance thin filament Ca2+-sensitization. Our goal here was to determine the structural basis for this enhancement by carrying out 3D reconstruction of thin filaments decorated with L348P-mutant cMyBP-C. When thin filaments were decorated with wild type N-terminal domains at low Ca2+, tropomyosin moved from the blocked to the closed position, as found previously. In contrast, the L348P mutant caused a significantly larger tropomyosin shift, to approximately the open position, consistent with its enhancement of Ca2+-sensitization. Phosphorylated wild type fragments showed a smaller shift than unphosphorylated fragments, whereas the shift induced by the L348P mutant was not affected by phosphorylation. We conclude that the L348P mutation causes a gain of function by enhancing tropomyosin displacement on the thin filament in a phosphorylation-independent way.

Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

Thermostability and reactivity in organic solvent of O-phospho-L-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1.

O-phospho-l-serine sulfhydrylase (OPSS) from archaeon Aeropyrum pernix K1 is able to synthesize l-cysteine even at 80 °C. In this article, we compared thermal stability and reactivity in organic solvent of OPSS with those of O-acetyl-l-serine sulfhydrylase B (OASS-B) from Escherichia coli. As a result, the thermostability of OPSS was much higher than that of OASS-B. Moreover, the activity of OPSS increased in the reaction mixture containing the organic solvent, such as N, N'-dimethyl formamide and 1,4-dioxane, whereas that of OASS-B gradually decreased as the content of organic solvent increased. From the crystal structural analysis, the intramolecular electrostatic interactions of N-terminal domain in OPSS seemed to be correlated with the tolerance of OPSS to high temperature and organic solvent. These results indicate that OPSS is more superior to OASS-B for the industrial production of l-cysteine and unnatural amino acids that are useful pharmaceuticals in the presence of organic solvent.