ABSTRACT
In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our in vitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with in vivo experiments in bacteria in the future.
Subject(s)
Bacterial Proteins , Catalytic Domain , Dinucleoside Phosphates , Models, Molecular , Streptococcus pneumoniae , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/metabolism , Crystallography, X-Ray , Dinucleoside Phosphates/metabolism , Dinucleoside Phosphates/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Substrate Specificity , Hydrolases/chemistry , Hydrolases/metabolism , Hydrolases/genetics , Protein Binding , Amino Acid Sequence , Hydrolysis , Binding SitesABSTRACT
Shugoshin-1 (Sgo1) is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis. It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120. However, it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis. Here, we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis. Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A. Interestingly, the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere, resulting in aberrant chromosome segregation and premature chromatid separation. Taken together, these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1-CENP-A interaction guides accurate cell division.
ABSTRACT
In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.
Subject(s)
CDC2 Protein Kinase , Chromosomal Proteins, Non-Histone , Chromosome Segregation , Humans , CDC2 Protein Kinase/metabolism , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Kinetochores/metabolism , Mitosis , PhosphorylationABSTRACT
BACKGROUND: Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy. METHODS: HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys. RESULTS: HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies. CONCLUSION: We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.
Subject(s)
Antibodies, Bispecific , Colorectal Neoplasms , Programmed Cell Death 1 Receptor , Animals , Humans , Mice , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Immunotherapy , Macaca fascicularis , Antibodies, Bispecific/pharmacologyABSTRACT
Bacteria could survive stresses by a poorly understood mechanism that contributes to the emergence of bacterial persisters exhibiting multidrug tolerance (MDT). Recently, Pseudoalteromonas rubra prpAT module was found to encode a toxin PrpT and corresponding cognate antidote PrpA. In this study, we first reported multiple individual and complex structures of PrpA and PrpT, which uncovered the high-resolution three-dimensional structure of the PrpT:PrpA2:PrpT heterotetramer with the aid of size exclusion chromatography-multi-angle light scattering experiments (SEC-MALS). PrpT:PrpA2:PrpT is composed of a PrpA homodimer and two PrpT monomers which are relatively isolated from each other and from ParE family. The superposition of antitoxin monomer structures from these structures highlighted the flexible C-terminal domain (CTD). A striking conformational change in the CTDs of PrpA homodimer depolymerized from homotetramer was provoked upon PrpT binding, which accounts for the unique PrpT-PrpARHH mutual interactions and further neutralizes the toxin PrpT. PrpA2-54-form I and II crystal structures both contain a doughnut-shaped hexadecamer formed by eight homodimers organized in a cogwheel-like form via inter-dimer interface dominated by salt bridges and hydrogen bonds. Moreover, PrpA tends to exist in solution as a homodimer other than a homotetramer (SEC-MALS) in the absence of flexible CTD. Multiple multi-dimers, tetramer and hexamer included, of PrpA2-54 mediated by the symmetric homodimer interface and the complicated inter-dimer interface could be observed in the solution. SEC-MALS assays highlighted that phosphate buffer (PB) and the increase in the concentration appear to be favorable for the PrpA2-54 oligomerization in the solution. Taken together with previous research, a model of PrpA2-54 homotetramer in complex with prpAT promoter and the improved mechanism underlying how PrpTA controls the plasmid replication were proposed here.
ABSTRACT
Gene expression is mediated by a series of regulatory proteins, i.e., transcription factors. Under different growth conditions, the transcriptional regulation of structural genes is associated with the recognition of specific regulatory elements (REs) in promoter DNA. The manner by which transcription factors recognize distinctive REs is a key question in structural biology. Previous research has demonstrated that Ino2p/Ino4p heterodimer is associated with the transcriptional regulation of phospholipid biosynthetic genes. Mechanistically, Ino2p/Ino4p could specifically recognize the inositol/choline-responsive element (ICRE), followed by the transcription activation of the phospholipid biosynthetic gene. While the promoter DNA sequence for Ino2p has already been characterized, the structural basis for the mutual interaction between Ino2p/Ino4p and their binding interface with promoter DNA remain relatively unexplored. Here, we have determined the crystalline structure of the Ino2pDBD/Ino4pDBD/DNA ternary complex, which highlights some residues (Ino2pHis12/Glu16/Arg20/Arg44 and Ino4pHis12/Glu16/Arg19/Arg20) associated with the sequence-specific recognition of promoter DNA. Our biochemical analysis showed that mutating these residues could completely abolish protein-DNA interaction. Despite the requirement of Ino2p and Ino4p for interprotein-DNA interaction, both proteins can still interact-even in the absence of DNA. Combined with the structural analysis, our in vitro binding analysis demonstrated that residues (Arg35, Asn65, and Gln69 of Ino2pDBD and Leu59 of Ino4pDBD) are critical for interprotein interactions. Together, these results have led to the conclusion that these residues are critical to establishing interprotein-DNA and protein-DNA mutual interactions.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Saccharomyces cerevisiae Proteins , Transcription Factors , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA/genetics , DNA/metabolism , Gene Expression Regulation, Fungal/genetics , Gene Expression Regulation, Fungal/physiology , Phospholipids/metabolism , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
PCI domain proteins play important roles in post-transcriptional gene regulation. In the TREX-2 complex, PCI domain-containing Sac3 and Thp1 proteins and accessory Sem1 protein form a ternary complex required for mRNA nuclear export. In contrast, structurally related Thp3-Csn12-Sem1 complex mediates pre-mRNA splicing. In this study, we determined the structure of yeast Thp3186-470-Csn12-Sem1 ternary complex at 2.9 Å resolution. Both Thp3 and Csn12 structures have a typical PCI structural fold, characterized by a stack of α-helices capped by a C-terminal winged-helix (WH) domain. The overall structure of Thp3186-470-Csn12-Sem1 complex has an inverted V-shape with Thp3 and Csn12 forming the two sides. A fishhook-shaped Sem1 makes extensive contacts on Csn12 to stabilize its conformation. The overall structure of Thp3186-470-Csn12-Sem1 complex resembles the previously reported Sac3-Thp1-Sem1 complex, but also has significant structural differences. The C-terminal WH domains of Thp3 and Csn12 form a continuous surface to bind different forms of nucleic acids with micromolar affinity. Mutation of the basic residues in the WH domains of Thp3 and Csn12 affects nucleic acid binding in vitro and mRNA splicing in vivo. The Thp3-Csn12-Sem1 structure provides a foundation for further exploring the structural elements required for its specific recruitment to spliceosome for pre-mRNA splicing.
Subject(s)
Percutaneous Coronary Intervention , Saccharomyces cerevisiae Proteins , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA, Messenger/genetics , RNA, Messenger/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
A new software package, autoPX, for processing X-ray diffraction data from biomacromolecular crystals is reported. This processing software package is designed on the basis of novel methods such as the location of diffraction spots by an improved Canny operator, indexing by a modified Fourier transform, a novel definition of mosaicity that expresses the dispersion state of reciprocal diffraction spots, and the correction of predicted diffraction spot coordinates by homography transform. New programming of some traditional algorithms necessary for integration and scaling is also included. Several examples of crystal structure determination using data from the SSRF beamlines reduced using autoPX, HKL-2000, DIALS and XDS are also demonstrated, and indicate that autoPX is capable of processing diffraction data from biomacromolecular crystals and providing adequate solutions to problems encountered at the SSRF beamlines.
Subject(s)
Algorithms , Synchrotrons , Software , X-Ray DiffractionABSTRACT
Uncovering conserved 3D protein-ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein-ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG-motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.
Subject(s)
Proteins , Ligands , Proteins/metabolism , Protein Binding , Binding SitesABSTRACT
Toxin-antitoxin (TA) systems are ubiquitous regulatory modules for bacterial growth and cell survival following stress. YefM-YoeB, the most prevalent type II TA system, is present in a variety of bacterial species. In Staphylococcus aureus, the YefM-YoeB system exists as two independent paralogous copies. Our previous research resolved crystal structures of the two oligomeric states (heterotetramer and heterohexamer-DNA ternary complex) of the first paralog as well as the molecular mechanism of transcriptional autoregulation of this module. However, structural details reflecting molecular diversity in both paralogs have been relatively unexplored. To understand the molecular mechanism of how Sa2YoeB and Sa2YefM regulate their own transcription and how each paralog functions independently, we solved a series of crystal structures of the Sa2YoeB-Sa2YefM. Our structural and biochemical data demonstrated that both paralogous copies adopt similar mechanisms of transcriptional autoregulation. In addition, structural analysis suggested that molecular diversity between the two paralogs might be reflected in the interaction profile of YefM and YoeB and the recognition pattern of promoter DNA by YefM. Interaction analysis revealed unique conformational and activating force effected by the interface between Sa2YoeB and Sa2YefM. In addition, the recognition pattern analysis demonstrated that residues Thr7 and Tyr14 of Sa2YefM specifically recognizes the flanking sequences (G and C) of the promoter DNA. Together, these results provide the structural insights into the molecular diversity and independent function of the paralogous copies of the YoeB-YefM TA system.
Subject(s)
Antitoxins , Bacterial Toxins , DNA, Bacterial , Staphylococcus aureus , Toxin-Antitoxin Systems , Antitoxins/chemistry , Antitoxins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , DNA, Bacterial/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolismABSTRACT
TYE7, a bHLH (basic helix-loop-helix) transcription factor from Saccharomyces cerevisiae, is involved in the regulation of many genes, including glycolytic genes. Meanwhile, accumulating evidence indicates that TYE7 also functions as a cyclin and is linked to sulfur metabolism. Here, the structure of TYE7 (residues 165-291) complexed with its specific DNA was determined by X-ray crystallography. Structural analysis and comparison revealed that His185 and Glu189 are conserved in base recognition. However, Arg193 is also involved in base recognition in the structures that were compared. In the structure in this study, Arg193 in chain A has two conformations and makes a salt bridge with the phosphate backbone structure. In addition, a series of corresponding electrophoretic mobility shift assays were performed to better understand the DNA-binding mechanism of the bHLH domain of TYE7.
Subject(s)
Crystallography, X-Ray/methods , DNA, Fungal/chemistry , DNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Amino Acid Sequence , Binding Sites , Models, Molecular , Protein Conformation , Sequence HomologyABSTRACT
Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the transfer of the phosphoribosyl group of 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to anthranilate to form phosphoribosyl-anthranilate. Crystal structures of AnPRTs from bacteria and archaea have previously been determined; however, the structure of Saccharomyces cerevisiae AnPRT (ScAnPRT) still remains unsolved. Here, crystal structures of ScAnPRT in the apo form as well as in complex with its substrate PRPP and the substrate analogue 4-fluoroanthranilate (4FA) are presented. These structures demonstrate that ScAnPRT exhibits the conserved structural fold of type III phosphoribosyltransferase enzymes and shares the similar mode of substrate binding found across the AnPRT protein family. In addition, crystal structures of ScAnPRT mutants (ScAnPRTSer121Ala and ScAnPRTGly141Asn) were also determined. These structures suggested that the conserved residue Ser121 is critical for binding PRPP, while Gly141 is dispensable for binding 4FA. In summary, these structures improved the preliminary understanding of the substrate-binding mode of ScAnPRT and laid foundations for future research.
Subject(s)
Anthranilate Phosphoribosyltransferase/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Diphosphates/metabolism , Protein Multimerization , Protein Structure, Secondary , ortho-Aminobenzoates/metabolismABSTRACT
Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "ß-switch" region through the ß chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.
Subject(s)
Crotalid Venoms/metabolism , Fibrinolytic Agents/metabolism , Lectins, C-Type/metabolism , Platelet Glycoprotein GPIb-IX Complex/chemistry , Crystallography, X-Ray , Humans , Immunoprecipitation , Models, Molecular , Platelet Glycoprotein GPIb-IX Complex/metabolism , Protein Binding/drug effects , Protein Conformation , Protein Domains , Protein Interaction Mapping , Structure-Activity Relationship , Surface Plasmon Resonance , Thrombin/metabolism , von Willebrand Factor/metabolismABSTRACT
YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.
Subject(s)
Bacterial Proteins/ultrastructure , Endoribonucleases/ultrastructure , Escherichia coli Proteins/genetics , Staphylococcus aureus/ultrastructure , Toxin-Antitoxin Systems/genetics , Antitoxins/genetics , Antitoxins/ultrastructure , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , DNA-Binding Proteins/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/ultrastructure , Operon/genetics , Promoter Regions, Genetic , Protein Binding/genetics , Protein Multimerization/genetics , Staphylococcus aureus/chemistry , Staphylococcus aureus/geneticsABSTRACT
Molecular chaperones can prevent and repair protein misfolding and aggregation to maintain protein homeostasis in cells. Hsp40 chaperones interact with unfolded client proteins via the dynamic multivalent interaction (DMI) mechanism with their multiple client-binding sites. Here we report that a type I Hsp40 chaperone from Streptococcus pneumonia (spHsp40) forms a concentration-independent polydispersity oligomer state in solution. The crystal structure of spHsp40 determined at 2.75 Å revealed that each monomer has a type I Hsp40 structural fold containing a zinc finger domain and C-terminal domains I and II (CTD I and CTD II). Subsequent quaternary structure analysis using a PISA server generated two dimeric models. The interface mutational analysis suggests the conserved C-terminal dimeric motif as a basis for dimer formation and that the novel dimeric interaction between a client-binding site in CTD I and the zinc finger domain promotes the formation of the spHsp40 oligomeric state. In vitro functional analysis demonstrated that spHsp40 oligomer is fully active and possess the optimal activity in stimulating the ATPase activity of spHsp70. The oligomer state of type I Hsp40 and its formation might be important in understanding Hsp40 function and its interaction with client proteins.
Subject(s)
Bacterial Proteins/chemistry , HSP40 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/chemistry , Streptococcus pneumoniae/chemistry , Crystallography, X-Ray , Protein Domains , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal, Humanized/chemistry , Antigen-Antibody Complex/chemistry , Antineoplastic Agents, Immunological/chemistry , Neoplasms/therapy , Receptor, ErbB-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Binding Sites, Antibody , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Crystallization , Crystallography, X-Ray/methods , Female , Humans , Protein Domains , Receptor, ErbB-2/immunologyABSTRACT
Phosphopantothenoylcysteine (PPC) synthetase (PPCS) catalyzes nucleoside triphosphate-dependent condensation reaction between 4'-phosphopantothenate (PPA) and l-cysteine to form PPC in CoA biosynthesis. The catalytic mechanism of PPCS has not been resolved yet. Coenzyme A biosynthesis protein 2 (Cab2) possesses activity of PPCS in Saccharomyces cerevisiae. Our enzymatic assays suggest that Cab2 could utilize both ATP and CTP to activate PPA in vitro. The results of isothermal titration calorimetry indicate that PPA, CTP, and ATP could bind to Cab2 individually, with PPA having the highest binding affinity. To provide further insight into the catalytic mechanism of Cab2, we determined the crystal structures of Cab2 and its complex with PPA, the reaction intermediate 4'-phosphopantothenoyl-CMP, the final reaction product PPC, and the product analogue phosphopantothenoylcystine. Except for PPA, all other ligands were generated in situ and present in the active-site pocket of Cab2. Structures of Cab2 in complex with ligands provide insight into substrates binding and its catalytic mechanism. Analysis of structures indicates that the carboxyl of PPA-moiety of ligands and the γ-amino group of Asn97 possess different conformations in these complex structures. The cysteine/cystine/serine selectivity assays for Cab2 indicate that the amino group rather than the thiol group of l-cysteine attacks the carbonyl of 4'-phosphopantothenoyl-CMP to form PPC. Based on structural and biochemical data, the catalytic mechanism of Cab2 was proposed for the first time.
Subject(s)
Peptide Synthases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Catalysis , Crystallography/methods , Protein Binding/physiologyABSTRACT
Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.