Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Radiographic changes and factors associated with subsequent progression of damage in weight-bearing joints of patients with rheumatoid arthritis under TNF-blocking therapies-three-year observational study.

OBJECTIVES: The long-term effects of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis (RA) have not been fully characterized. The purpose of this study was to assess the radiographic changes of weight-bearing joints in patients with RA during 3-year of TNF-blocking therapies and to identify factors related to the progression of joint damage. METHODS: Changes in clinical variables and radiological findings in 243 weight-bearing joints (63 hips, 54 knees, 71 ankles, and 55 subtalar joints) in 38 consecutive patients were investigated during three years of treatment with TNF-blocking agents. Multivariate logistic regression analysis was used to identify risk factors for the progression of weight-bearing joint damage. RESULTS: Seventeen (14.5%) of proximal weight-bearing joints (hips and knees) showed apparent radiographic progression during three years of treatment, whereas none of the proximal weight-bearing joints showed radiographic evidence of improvement or repair. In contrast, distal weight-bearing joints (ankle and subtalar joints) displayed radiographic progression and improvement in 20 (15.9%) and 8 (6.3%) joints, respectively. Multivariate logistic analysis for proximal weight-bearing joints identified the baseline Larsen grade (p < 0.001, OR:24.85, 95%CI: 5.07-121.79) and disease activity at one year after treatment (p = 0.003, OR:3.34, 95%CI:1.50-7.46) as independent factors associated with the progression of joint damage. On the other hand, multivariate analysis for distal weight-bearing joints identified disease activity at one year after treatment (p < 0.001, OR:2.13, 95%CI:1.43-3.18) as an independent factor related to the progression of damage. CONCLUSIONS: Baseline Larsen grade was strongly associated with the progression of damage in the proximal weight-bearing joints. Disease activity after treatment was an independent factor for progression of damage in proximal and distal weight-bearing joints. Early treatment with TNF-blocking agents and tight control of disease activity are necessary to prevent the progression of damage of the weight-bearing joints.

Clinical features and prognosis of conjunctival melanoma in Japanese patients.

PURPOSE: To evaluate the clinical features and prognosis of conjunctival melanoma in Japanese patients. STUDY DESIGN: Retrospective observational case series. METHODS: Twenty patients (8 men and 12 women) diagnosed with conjunctival melanoma at a singlehospital between 2003 and 2017 were analyzed. Data on clinical presentation, sex, age, the affected eye, tumor location, tumor origin, tumor stage according to the American Joint Committee on Cancer staging system (eighth edition), treatment, outcomes, local recurrence, metastasis, and survival were extracted from the patients' medical records and reviewed. RESULTS: The mean age at diagnosis was 64.2 ± 14.8 years. Tumor locations at the first examination included the bulbar conjunctiva (n = 19), plica (n = 13), and fornix (n = 12). The tumor stage was T1 in 5 cases (25%), T2 in 12 cases (60%), T3 in 3 cases (15%), and T4 in none. The mean follow-up duration was 91.7 ± 46.0 months. The local recurrence rates at 1, 5, and 10 years were 5.0%, 18.8%, and 31.5%, respectively, whilst the metastasis rates were 5.0%, 25.6%, and 32.4%, respectively. Four of the 6 patients who experienced metastasis died; duration from metastasis to death was 17.5 months (range, 7-25). The 5-year survival rate for conjunctival melanoma was 78.8%. Tumor thickness was significantly associated with survival duration on univariate Cox regression analyses. CONCLUSION: The mortality rate for conjunctival melanoma in the Japanese population was lower and higher than that reported in the Chinese and United States populations, respectively. Tumor thickness was a prognostic factor for survival in patients with conjunctival melanoma.

Risk factors for myopia at 1-year corrected age following laser photocoagulation for retinopathy of prematurity.

BACKGROUND/OBJECTIVES: The prevalence of myopia is higher in preterm infants who underwent laser photocoagulation (LPC) for retinopathy of prematurity (ROP). The aim of this study was to investigate factors associated with myopia in preterm infants who undergo LPC for ROP. SUBJECTS/METHODS: We retrospectively analysed the medical records of preterm infants born at Kyushu University Hospital (October 2008-March 2018) at ≤32 weeks of gestational age or with birth weight ≤1500 g. We evaluated the associations between nine clinical factors and the spherical equivalent at 1-year corrected age by performing multivariable linear regression in LPC-treated ROP patients. RESULTS: Among the 485 infants enroled, 76 developed ROP requiring treatment. Of these, 71 underwent LPC, which was provided to 63 infants as the primary treatment (LPC alone or the combination therapy of LPC and intravitreal injection of bevacizumab [IVB]) and to eight infants as additional LPC after IVB monotherapy. The results of a refractive examination at 1-year corrected age were available for 110 eyes of 56 infants (78.9%). The mean ± standard deviation of the SE value was -0.5 ± 3.0 dioptres (D). Multivariable linear regression analysis revealed a significant association between laser spot count and SE value (ß = -0.081 ± 0.040 D per 100 spots [mean ± standard error], p = 0.045). CONCLUSIONS: Our results suggest that an increased laser spot count observed during ROP treatment associates with myopia.

Rheumatoid wrist deformity and risk of extensor tendon rupture evaluated by 3DCT imaging.

OBJECTIVE: Extensor tendon rupture on the dorsum of the wrist is commonly seen in patients with rheumatoid arthritis (RA). It causes immediate dysfunction of the hand and surgical reconstruction is usually required. The purpose of this study was to clarify the risk of extensor tendon rupture by quantifying wrist deformity on three-dimensional computed tomography (3DCT) images. MATERIALS AND METHODS: Three-dimensional CT images of 108 wrists in 102 patients with RA and 38 wrists in 38 healthy volunteers were analyzed retrospectively. All of the rheumatoid wrists had caused persistent pain for more than 6 months despite ongoing medical treatment. Extensor tendon rupture was noted in 49 wrists in 47 patients, and no rupture was noted in 59 wrists in 56 patients. The dorsal subluxation ratio (DSR) of the ulnar head and the carpal supination angle (CSA) were measured utilizing a new technique. RESULTS: The average DSR and CSA in the rupture group (n = 49), the non-rupture group (n = 59), and the normal wrist group (n = 38) were 37%, 19%, and 26%, and 15 degrees , 11 degrees , and 6 degrees respectively. The cut-off values for extensor tendon rupture in the wrists of patients with RA were 32% (sensitivity; 70%, specificity; 75%) in the DSR, and 14 degrees (71%, 68%) in the CSA. CONCLUSION: By utilizing 3DCT imaging of the rheumatoid wrist, these parameters can help improve our ability to predict extensor tendon rupture.

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of M. mazei PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCO*Lys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.

Neurodevelopmental outcomes following intravitreal bevacizumab injection in Japanese preterm infants with type 1 retinopathy of prematurity.

PURPOSE: The purpose of this study was to evaluate neurodevelopmental outcomes in 18-month old (corrected age) preterm infants who received an intravitreal bevacizumab (IVB) injection for the treatment of type 1 retinopathy of prematurity (ROP). METHODS: In this ten-year retrospective study, we reviewed the medical records of patients who underwent ROP screening at Kyushu University Hospital. Among the patients who received IVB or laser photocoagulation (LPC) for the treatment of type 1 ROP, we included infants whose neurodevelopmental examination (the Kyoto Scale of Psychological Development [KSPD]) results at 18 months corrected age were available. Then, the effect of IVB on the developmental quotient (DQ) in each KSPD domain (Postural-Movement, Cognitive-Adaptive, or Language-Social domain) or the overall DQ was investigated by performing linear regression analysis. RESULTS: Out of the 513 patients reviewed, 53 were included in the study. IVB and LPC were performed for 14 and 39 patients, respectively. Administration of IVB was significantly associated with neurodevelopmental delay in the Language-Social domain (p = 0.01). The observed association remained even after adjusting for gestational age and birth weight (p = 0.03). CONCLUSIONS: Administration of IVB may introduce a risk of developmental impairment of interpersonal relationships, socializations, and/or verbal abilities of preterm children. We recommended that preterm infants who received IVB undergo a neurodevelopmental reassessment during their school years or in adulthood.

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Structural and functional differences of SWIRM domain subtypes.

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.

Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation.

Cell-free protein synthesis has become one of the standard methods for protein expression. One of the major advantages of this method is that PCR-amplified linear DNA fragments can be directly used as templates for protein synthesis. The productivity of cell-free protein synthesis using linear DNA templates is generally lower than that from plasmid DNA templates, especially when using an Escherichia coli cell extract. In the present study, we found that a simple modification of the protocol for cell extract preparation from E. coli, just by altering the cultivation temperature (37 degrees C) of the cells to a moderately lower range (20-34 degrees C), dramatically reduced the linear DNA degradation activity in the cell extract. This modification greatly improved the productivity of cell-free protein synthesis from linear DNA templates. The removal of the RecD protein, one of the components of exonuclease V, from the extract had almost the same effect, indicating that the linear DNA degradation activity in the extract was mainly due to the RecD protein and that its expression level was decreased at the lower cultivation temperature.

Solution structure of the SWIRM domain of human histone demethylase LSD1.

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.

Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.

Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using the Escherichia coli cell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs from Methanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Solution structure of the general transcription factor 2I domain in mouse TFII-I protein.

The general transcription factor TFII-I, with the corresponding gene name GTF2I, is an unusual transcriptional regulator that associates with both basal and signal-induced transcription factors. TFII-I consists of six GTF2I repeat domains, called I-repeats R1-R6. The structure and function of the GTF2I domain are not clearly understood, even though it contains a helix-loop-helix motif, which is considered to be the protein-protein interaction area, based on biochemical analyses. Here, we report the solution structure of the fifth repeat of the six GTF2I repeat domains from murine TFII-I, which was determined by heteronuclear multidimensional NMR spectroscopy (PDB code 1Q60). The three-dimensional structure of the GTF2I domain is classified as a new fold, consisting of four helices (residues 8-24, 34-39, 63-71, and 83-91), two antiparallel beta strands (residues 44-47 and 77-80), and a well-defined loop containing two beta-turns between sheet 1 and helix 3. All of the repeats probably have similar folds to that of repeat 5, because the conserved residues in the GTF2I repeat domains are assembled on the hydrophobic core, turns, and secondary structure elements, as revealed by a comparison of the sequences of the first through the sixth GTF2I repeats in TFII-I.

Solution structure of the zinc finger HIT domain in protein FON.

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3.

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure.

SQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of approximately 80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove.

Solution structure of the major DNA-binding domain of Arabidopsis thaliana ethylene-insensitive3-like3.

Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five alpha-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding.

The CAP-Gly domain of CYLD associates with the proline-rich sequence in NEMO/IKKgamma.

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain. Here we report that the third CAP-Gly domain of CYLD specifically interacts with one of the two proline-rich sequences of NEMO/IKKgamma. The tertiary structure of the CAP-Gly domain shares the five-stranded beta sheet topology with the SH3 domain, which is well known as a proline-rich sequence-recognition domain. However, chemical shift mapping revealed that the peptide binding site of the CAP-Gly domain is formed without the long peptide binding loop characteristic of the SH3 domain. Therefore, CAP-Gly is likely to be a novel proline-rich sequence binding domain with a mechanism different from that of the SH3 domain.