Assembly of The Mitochondrial Complex†I Assembly Complex Suggests a Regulatory Role for Deflavination.

Fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are mitochondrial redox processes that generate ATP. The biogenesis of the respiratory Complex I, a 1 MDa multiprotein complex that is responsible for initiating OXPHOS, is mediated by assembly factors including the mitochondrial complex I assembly (MCIA) complex. However, the organisation and the role of the MCIA complex are still unclear. Here we show that ECSIT functions as the bridging node of the MCIA core complex. Furthermore, cryo-electron microscopy together with biochemical and biophysical experiments reveal that the C-terminal domain of ECSIT directly binds to the vestigial dehydrogenase domain of the FAO enzyme ACAD9 and induces its deflavination, switching ACAD9 from its role in FAO to an MCIA factor. These findings provide the structural basis for the MCIA complex architecture and suggest a unique molecular mechanism for coordinating the regulation of the FAO and OXPHOS pathways to ensure an efficient energy production.

High-frequency Ultrasound Assessment of Systemic Sclerosis Skin Involvement: Intraobserver Repeatability and Relationship With Clinician Assessment and Dermal Collagen Content.

OBJECTIVE: The modified Rodnan skin score (mRSS) remains the preferred method for skin assessment in systemic sclerosis (SSc). There are concerns regarding high interobserver variability of mRSS and negative clinical trials utilizing mRSS as the primary endpoint. High-frequency ultrasound (HFUS) allows objective assessment of cutaneous fibrosis in SSc. We investigated the relationship between HFUS with both mRSS and dermal collagen. METHODS: Skin thickness (ST), echogenicity, and novel shear wave elastography (SWE) were assessed in 53 patients with SSc and 15 healthy controls (HCs) at the finger, hand, forearm, and abdomen. The relationship between HFUS parameters with mRSS (n = 53) and dermal collagen (10 patients with SSc and 10 HCs) was investigated. Intraobserver repeatability of HFUS was calculated using intraclass correlation coefficients (ICCs). RESULTS: HFUS assessment of ST (hand/forearm) and SWE (finger/hand) correlated with local mRSS at some sites. Subclinical abnormalities in ST, echogenicity, and SWE were present in clinically uninvolved SSc skin. Additionally, changes in echogenicity and SWE were sometimes apparent despite objectively normal ST on HFUS. ST, SWE, and local mRSS correlated strongly with collagen quantification (r = 0.697, 0.709, 0.649, respectively). Intraobserver repeatability was high for all HFUS parameters (ICCs for ST = 0.946-0.978; echogenicity = 0.648-0.865; and SWE = 0.953-0.973). CONCLUSION: Our data demonstrate excellent reproducibility and reassuring convergent validity with dermal collagen content. Detection of subclinical abnormalities is an additional benefit of HFUS. The observed correlations with collagen quantification support further investigation of HFUS as an alternative to mRSS in clinical trial settings.

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A.

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.

Auxin sensing is a property of an unstructured domain in the Auxin Response Factor ETTIN of Arabidopsis thaliana.

The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations.

We have constructed novel plasmids pANY2, pANY3, and pANY6 for flexible cloning with low false positives, efficient expression, and convenient purification of proteins. The pANY2 plasmid can be used for efficient isopropyl-ß-d-thiogalactoside (IPTG) induced protein expression, while the pANY3 plasmid can be used for temperature-induced expression. The pANY6 plasmid contains a self-cleaving elastin-like protein (ELP) tag for purification of recombinant protein by simple ELP-mediated precipitation steps and removal of the ELP tag by self-cleavage. A urea-based denaturation and refolding processes for renaturation of insoluble inclusion bodies can be conveniently integrated into the ELP-mediated precipitation protocol, removing time-consuming dialysis steps. These novel vectors, together with the described strategies of gene cloning, protein expression, and purification, may have wide applications in biosciences, agricultural, food technologies, and so forth.

A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc.

The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding.

Vaccinia virus (VACV), the prototype member of the Poxviridae, replicates in the cytoplasm of an infected cell. The catalytic subunit of the DNA polymerase E9 binds the heterodimeric processivity factor A20/D4 to form the functional polymerase holoenzyme. Here we present the crystal structure of full-length E9 at 2.7 Å resolution that permits identification of important poxvirus-specific structural insertions. One insertion in the palm domain interacts with C-terminal residues of A20 and thus serves as the processivity factor-binding site. This is in strong contrast to all other family B polymerases that bind their co-factors at the C terminus of the thumb domain. The VACV E9 structure also permits rationalization of polymerase inhibitor resistance mutations when compared with the closely related eukaryotic polymerase delta-DNA complex.

Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7.

The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255-1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.

ESPRIT: A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences.

Production of soluble, purifiable domains or multi-domain fragments of proteins is a prerequisite for structural biology and other applications. When target sequences are poorly annotated, or when there are few similar sequences available for alignments, identification of domains can be problematic. A method called expression of soluble proteins by random incremental truncation (ESPRIT) addresses this problem by high-throughput automated screening of tens of thousands of enzymatically truncated gene fragments. Rare soluble constructs are identified by experimental screening, and the boundaries revealed by DNA sequencing.

Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1).

Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.

Structural insights into the architecture of the Shigella flexneri virulence factor IcsA/VirG and motifs involved in polar distribution and secretion.

IcsA/VirG is a key virulence factor of the human pathogen Shigella flexneri, acting as both an adhesin and actin-polymerizing factor during infection. We identified a soluble expression construct of the IcsA/VirG α-domain using the ESPRIT library screening system and determined its structure to 1.9Å resolution. In addition to the previously characterized autochaperone domain, our structure reveals a new domain, which shares a common fold with the autochaperone domains of various autotransporters. We further provide insight into the previously structurally uncharacterized ß-helix domain that harbors the polar targeting motif and passenger-associated transport repeat. This structure is the first of any member of the recently identified passenger-associated transport repeat-containing autotransporters. Thus, it provides new insights into the overall architecture of this class of autotransporters, the function of the identified additional autochaperone domain and the structural properties of motifs involved in polar targeting and secretion of the Shigella flexneri virulence factor IcsA/VirG.

A structural organization for the Disrupted in Schizophrenia 1 protein, identified by high-throughput screening, reveals distinctly folded regions, which are bisected by mental illness-related mutations.

Disrupted in Schizophrenia 1 (DISC1) is a scaffolding protein of significant importance for neurodevelopment and a prominent candidate protein in the pathology of major mental illness. DISC1 modulates a number of critical neuronal signaling pathways through protein-protein interactions; however, the mechanism by which this occurs and how DISC1 causes mental illness is unclear, partly because knowledge of the structure of DISC1 is lacking. A lack of homology with known proteins has hindered attempts to define its domain composition. Here, we employed the high-throughput Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT) technique to identify discretely folded regions of human DISC1 via solubility assessment of tens of thousands of fragments of recombinant DISC1. We identified four novel structured regions, named D, I, S, and C, at amino acids 257-383, 539-655, 635-738, and 691-836, respectively. One region (D) is located in a DISC1 section previously predicted to be unstructured. All regions encompass coiled-coil or α-helical structures, and three are involved in DISC1 oligomerization. Crucially, three of these domains would be lost or disrupted by a chromosomal translocation event after amino acid 597, which has been strongly linked to major mental illness. Furthermore, we observed that a known illness-related frameshift mutation after amino acid 807 causes the C region to form aberrantly multimeric and aggregated complexes with an unstable secondary structure. This newly revealed domain architecture of DISC1, therefore, provides a powerful framework for understanding the critical role of this protein in a variety of devastating mental illnesses.

Expression and Characterization of Levansucrase from Clostridium acetobutylicum.

The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag+ and Hg2+. The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 µmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions.

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Recent advances in universal TA cloning methods for use in function studies.

As one of the simplest and most efficient cloning methods, T-vector-based TA cloning has been widely used for cloning of single genes and construction of DNA libraries. This approach is especially suitable for high-throughput cloning of diverse DNA fragments since inserts can be cloned without knowledge of their sequence; it is therefore an ideal tool for high-throughput analysis of protein structure and function. Although most of the currently available T-vectors can only be used for cloning purposes, some novel variants with improved functions have be developed. This review focuses on recent developments of universal TA cloning methods and T-vectors constructed for function studies.

Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites.

The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Što bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Use of Laser Speckle Contrast Imaging to Assess Digital Microvascular Function in Primary Raynaud Phenomenon and Systemic Sclerosis: A Comparison Using the Raynaud Condition Score Diary.

OBJECTIVE: Evaluate objective assessment of digital microvascular function using laser speckle contrast imaging (LSCI) in a cross-sectional study of patients with primary Raynaud phenomenon (RP) and systemic sclerosis (SSc), comparing LSCI with both infrared thermography (IRT) and subjective assessment using the Raynaud Condition Score (RCS) diary. METHODS: Patients with SSc (n = 25) and primary RP (n = 18) underwent simultaneous assessment of digital perfusion using LSCI and IRT with a cold challenge on 2 occasions, 2 weeks apart. The RCS diary was completed between assessments. The relationship between objective and subjective assessments of RP was evaluated. Reproducibility of LSCI/IRT was assessed, along with differences between primary RP and SSc, and the effect of sex. RESULTS: There was moderate-to-good correlation between LSCI and IRT (Spearman rho 0.58-0.84, p < 0.01), but poor correlation between objective assessments and the RCS diary (p > 0.05 for all analyses). Reproducibility of IRT and LSCI was moderate at baseline (ICC 0.51-0.63) and immediately following cold challenge (ICC 0.56-0.86), but lower during reperfusion (ICC 0.3-0.7). Neither subjective nor objective assessments differentiated between primary RP and SSc. Men reported lower median daily frequency of RP attacks (0.82 vs 1.93, p = 0.03). Perfusion using LSCI/IRT was higher in men for the majority of assessments. CONCLUSION: Objective and subjective methods provide differing information on microvascular function in RP. There is good convergent validity of LSCI with IRT and acceptable reproducibility of both modalities. Neither subjective nor objective assessments could differentiate between primary RP and SSc. Influence of sex on subjective and objective assessment of RP warrants further evaluation.

Molecular determinants for nuclear import of influenza A PB2 by importin α isoforms 3 and 7.

Influenza A virus polymerase subunit PB2 is a major virulence determinant implicated in pathogenicity and host adaptation. During cross-species virus transfer from avian to mammalian cells, PB2 switches specificity from importin α3 to α7. This specificity is not recapitulated in vitro, where PB2 binds all importin α isoforms with comparably high affinity. In this study, we investigated the structure, conformational dynamics, and autoinhibition of importin α isoforms 1, 3, and 7 in complex with PB2. Our data suggest that association of PB2 with α3 and α7 is favored by reduced autoinhibition of these isoforms and by the unique structure of the nuclear localization signal (NLS) domain of PB2. We propose that by recruiting importin α3 or α7 in the absence of importin ß, PB2 reduces the complexity of adaptor-mediated import to a pseudo-bimolecular reaction, thereby acquiring a kinetic advantage over classical NLS cargos, which form an import complex only when importin α and ß are simultaneously available.