• BMB Reports
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• v.42 no.6
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• pp.350-355
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• 2009

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher $K_m$ for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids ($\Delta$18) and a truncated N-terminal half lacking its first 18 amino acids ($\Delta$18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using $^{18}F-FDG$. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal half.

• Journal of the Korean Magnetic Resonance Society
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• v.22 no.1
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• pp.18-25
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• 2018

Replication protein A (RPA) is an essential single-stranded DNA binding protein in DNA processing. It is known that N terminal domain of RPA70 (RPA70N) recruits various protein partners including damage-response proteins such as p53, ATRIP, Rad9, and MRE11. Although the common binding residues of RPA70N were revealed, dynamic properties of the protein are not studied yet. In this study, we measured $^{15}N$ relaxation parameters ($T_1,\;T_2$ and heteronuclear NOE) of human RPA70N and analyzed them using model-free analysis. Our data showed that the two loops near the binding site experience fast time scale motion while the binding site does not. It suggests that the protein binding surface of RPA70N is mostly rigid for minimizing entropy cost of binding and the loops can experience conformational changes.

• Molecules and Cells
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• v.44 no.3
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• pp.179-185
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• 2021

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 Å, b = 52.97 Å, and c = 60.61 Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1039-1042
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• 2009

RNA polymerase II C-terminal domain phosphatase 1 containing ubiquitin like domain (UBLCP1) has been identified as a regulatory molecule of RNA polymerase II. UBLCP1 consists of ubiquitin like domain (UBL) and phosphatase domain homologous with UDP and CTD phosphatase. UBLCP1 was cloned into the E.coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using both affinity and gel-filtration chromatography. Domains of UBLCP1 protein were successfully purified as 7 mg/500 mL (UBLCP1, 36.78 KDa), 32 mg/500 mL (UBL, 9 KDa) and 8 mg/500 mL (phosphatase domain, 25 KDa) yielded in LB medium, respectively. Isotope-labeled samples including triple-labeled ($^2H/^{15}N/^{13}C$) UBLCP1 were also prepared for hetero-nuclear NMR experiments. $^{15}N-^{1}H$ 2D-HSQC spectra of UBLCP1 suggest that both UBL and phosphatase domain are properly folded and structurally independent each other. These data will promise us further structural investigation of UBLCP1 by NMR spectroscopy and/or X-ray crystallography.

• Molecules and Cells
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• v.43 no.12
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• pp.1023-1034
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• 2020

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α'-chain (α'NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α'NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.56-65
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• 1992

The complete nucleotide sequence of the Bacillus circulans F-2 RSDA gene, coding for raw starch digesting a-amylase (RSDA), has been determined. The RSDA structure gene consists of an open reading frame of 2508 bp. Six bp upstream of the translational start codon of the RSDA is a typical gram-positive Shine-Dalgarno sequence and the RSDA encodes a preprotein of 836 amino acids with an Mr of 96, 727. The gene was expressed from its own regulatory region in E. coli and two putative consensus promoter sequences were identified upstream of a ribosome binding site and an ATG start codon. Confirmation of the nucleotide sequence was obtained and the signal peptide cleavage site was identified by comparing the predicted amino acid sequence with that derived by N-terminal analysis of the purified RSDA. The deduced N-terminal region of the RSDA conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete amino acid sequence was deduced and homology with other enzymes was compared. The results suggested that the Thr-Ser-rich hinge region and the non-catalytic domain are necessary for efficient adsorption onto raw substrates, and the catalytic domain (60 kDa) is necessary for the hydrolysis of substrates, as suggested in previous studies (8, 9).

• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.135-142
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• 1996

Antigenic domain of jai or surface protein (p30) of Toxoplosmc Sondii was analyzed after polymerase chain reaction (PCR) of its gene fragments. Hydrophilic or hydrophobic moiety of amino acid sequences were expressed as glutathione S-transferase (G57) fusion proteins. Fragments of p30 gene were as follows: 737, total p30 open reading frame (ORF) ; S28, total ORF excluding N-terminal signal sequence and C-terminal hydrophobic sequence; Al9, N-terminal 2/3 parts of A28; A19, N-terminal 2/3 of S28; P9, C-terminal 2/3 part of S28; Z9. middle 1/3 of S28; and 29, C-terminal 1/3 of S28. respectively. Primer of each fragment was synthesized to include clamp sequence of EcoR I restriction site. PCR amplified DNA was inserted info GST (26 kDa) expression vector, PGEX-47-1 to transform into Escheri,hia coei (.JM105 strain). G57 fusion proteins were expressed with IPTG induction as 63. 54, 45, 45, 35, 36. and 35 kDa proteins measured by SDS-PAGE. Each fusion protein was confirmed with G57 detection kit. Western blot analysis with the serum of a toxoplasmosis patient revealed antigenicity in proteins expressed by T37. S28, and Al9 but not those by Pl8. X9, Y10, and Z9. Antigenicity of p30 seems to be located either in N-terminal 115 part in the presence of middle 1/3 part or in the oligopeptides between margins of the first and second 1/3 parts.

• BMB Reports
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• v.40 no.1
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• pp.44-52
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• 2007

Kinesins, as a kind of microtubule-based motor proteins, have a conserved microtubule-binding site in their motor domain. Here we report that two homologous kinesins in Arabidopsis thaliana, KatB and KatC, contain a second microtubule-binding site in their tail domains. The prokaryotic-expressed N-terminal tail domain of the KatC heavy chain can bind to microtubules in an ATP-insensitive manner. To identify the precise region responsible for the binding, a serious of truncated KatC cDNAs encoding KatC N-terminal regions in different lengths, KatC1-128, KatC1-86, KatC1-73 and KatC1-63, fused to Histidine-tags, were expressed in E. coli and affinity-purified. Microtubule cosedimentation assays show that the site at amino acid residues 74-86 in KatC is important for microtubule-binding. By similarity, we obtained three different lengths of KatB N-terminal regions, KatB1-384, KatB1-77, and KatB1-63, and analyzed their microtubule-binding ability. Cosedimentation assays indicate that the KatB tail domain can also bind to microtubules at the same site as and in a similar manner to KatC. Fluorescence microscopic observations show that the microtubule-binding site at the tail domain of KatB or KatC can induce microtubules bundling only when the stalk domain is present. Through pull-down assays, we show that KatB1-385 and KatC1-394 are able to interact specifically with themselves and with each other in vitro. These findings are significant for identifying a previously uncharacterized microtubule-binding site in the two kinesin proteins, KatB and KatC, and the functional relations between them.

• BMB Reports
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• v.43 no.1
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• pp.1-8
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• 2010

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs.

• Molecules and Cells
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• v.37 no.7
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• pp.526-531
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• 2014

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 Nterminal fragment encompassing NLS in PARP-$1^{+/+}$ and PARP-$1^{-/-}$ mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-$1^{+/+}$ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-$1^{-/-}$ cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.