Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

Kamishikiryo, Jun; Fukuhara, Hideo; Okabe, Yuki; Kuroki, Kazuhiko; Maenaka, Katsumi

doi:10.1074/jbc.m110.214254

Cited by 44 publications

(66 citation statements)

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“…The LLT1 H176C mutant protein, hereafter simply designated as LLT1, was expressed in Escherichia coli as inclusion bodies and refolded by the dilution method, as reported previously [23]. After the refolding, the protein was concentrated and purified by size exclusion chromatography, as a monomer, where majority of the LLT1 was apparently eluted at 18kDa, slightly larger than monomer value (14 kDa) calculated from the amino acid sequence (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1C). Our previous study revealed that the wild-type LLT1 harboring His176 is not stable and easily aggregated, but the LLT1 H176C mutant is much more stable and soluble [23], and could be crystallized as described above. In the wild-type LLT1, the free Cys163 likely contributes to facilitate aggregation, possibly due to additional disulfide bond formation and/or exposure of hydrophobic region to outside, which would not occur on the cell surface.…”

Section: Overall Structure Of Llt1mentioning

confidence: 99%

“…The expression and purification of the LLT1 H176C mutant protein (amino acid residues Leu71-Val191) were performed as described previously [37]. The expression plasmid LLT1/pET22 was introduced into the E. coli strain BL21 (DE3) pLysS.…”

Section: Expression and Purification Of Llt1 Proteinsmentioning

confidence: 99%

“…The binding affinities between the ligands and the receptors were quite diverse. LLT1 and AICL bind their ligands, NKRP1A and NKp80, respectively, with low affinity (LLT1-NKRP1A: Kdß50 μM [23], AICL-NKp80: Kdß4 μM [14]), whereas NKp65 binds to KACL with significantly higher affinity (Kdß0.01 μM) [15]. Since these differences are strongly correlated with the recognition mechanisms, further structural studies at atomic resolution will be required for their clarification.…”

Section: Introductionmentioning

confidence: 99%

“…To understand the signaling by LLT1-NKRP1A in comparison with those by other CTLRs, we have investigated the molecular mechanism of LLT1 recognition by NKRP1A. We previously revealed the biophysical characteristics of the LLT1-NKRP1A interaction, including the kinetic and thermodynamic properties, by a surface plasmon resonance analysis [23]. Based on a broad mutagenesis study, we identified the binding sites for both LLT1 and NKRP1A as the membrane distal areas, which are also utilized for ligand binding by other CTLRs (e.g.…”

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confidence: 99%

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Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

et al. 2015

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Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended β-sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.Keywords: Cell surface receptor r Crystal structure r C-type lectin-like receptor r Natural killer cell r Protein-protein interactions Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Katsumi Maenaka e-mail: maenaka@pharm.hokudai.ac.jp * Equal contribution.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 1606 S. Kita et al. Eur. J. Immunol. 2015. 45: 1605-1613 Introduction Natural killer (NK) cells are a component of innate immunity, and display cytotoxic activities against tumor cells and virally infected cells [1,2]. The activities of NK cells are precisely regulated by activating and inhibitory signals, through diverse receptors on the cell surface [3][4][5]. These receptors are encoded in two genomic regions, the leukocyte receptor complex (chromosome 19 in human) [6] and the NK gene complex (chromosome 12 in human) [7,8]. The leukocyte receptor complex encodes the NK receptors of the immunoglobulin (Ig) superfamily, such as killer Ig-like receptors and leukocyte Ig-like receptors, whereas the NK gene complex region encodes the NK receptors of the C-type lectinlike receptors (CTLRs). The CTLRs are subdivided into two groups, according to their expression patterns. Killer cell lectin-like receptors (KLRs) are expressed in NK cells, and C-type lectin receptors (CLECs) are expressed in non-NK cells [9]. The ligands of KLRs are (1) major histocompatibility complex (MHC) class I molecules and relatives, and (2) CLECs. The former members include CD94, the NKG2 subfamily [10], and the Ly49 subfamily [11], and the latter members are the NK receptor-P1 (NKRP1) subfamily [12,13]. The NKRP1 su...

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Section: Resultsmentioning

confidence: 99%

Section: Overall Structure Of Llt1mentioning

confidence: 99%

Section: Expression and Purification Of Llt1 Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

et al. 2015

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Template‐based modeling of diverse protein interactions in CAPRI rounds 38‐45

et al. 2019

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Structures of proteins complexed with other proteins, peptides, or ligands are essential for investigation of molecular mechanisms. However, the experimental structures of protein complexes of interest are often not available. Therefore, computational methods are widely used to predict these structures, and, of those methods, template‐based modeling is the most successful. In the rounds 38‐45 of the Critical Assessment of PRediction of Interactions (CAPRI), we applied template‐based modeling for 9 of 11 protein‐protein and protein‐peptide interaction targets, resulting in medium and high‐quality models for six targets. For the protein‐oligosaccharide docking targets, we used constraints derived from template structures, and generated models of at least acceptable quality for most of the targets. Apparently, high flexibility of oligosaccharide molecules was the main cause preventing us from obtaining models of higher quality. We also participated in the CAPRI scoring challenge, the goal of which was to identify the highest quality models from a large pool of decoys. In this experiment, we tested VoroMQA, a scoring method based on interatomic contact areas. The results showed VoroMQA to be quite effective in scoring strongly binding and obligatory protein complexes, but less successful in the case of transient interactions. We extensively used manual intervention in both CAPRI modeling and scoring experiments. This oftentimes allowed us to select the correct templates from available alternatives and to limit the search space during the model scoring.

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Integrative modeling of protein‐protein interactions with pyDock for the new docking challenges

et al. 2019

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The seventh CAPRI edition imposed new challenges to the modeling of protein‐protein complexes, such as multimeric oligomerization, protein‐peptide, and protein‐oligosaccharide interactions. Many of the proposed targets needed the efficient integration of rigid‐body docking, template‐based modeling, flexible optimization, multiparametric scoring, and experimental restraints. This was especially relevant for the multimolecular assemblies proposed in the CASP12‐CAPRI37 and CASP13‐CAPRI46 joint rounds, which were described and evaluated elsewhere. Focusing on the purely CAPRI targets of this edition (rounds 38‐45), we have participated in all 17 assessed targets (considering heteromeric and homomeric interfaces in T125 as two separate targets) both as predictors and as scorers, by using integrative modeling based on our docking and scoring approaches: pyDock, IRaPPA, and LightDock. In the protein‐protein and protein‐peptide targets, we have also participated with our webserver (pyDockWeb). On these 17 CAPRI targets, we submitted acceptable models (or better) within our top 10 models for 10 targets as predictors, 13 targets as scorers, and 4 targets as servers. In summary, our participation in this CAPRI edition confirmed the capabilities of pyDock for the scoring of docking models, increasingly used within the context of integrative modeling of protein interactions and multimeric assemblies.

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Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

Cited by 44 publications

References 33 publications

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

Template‐based modeling of diverse protein interactions in CAPRI rounds 38‐45

Integrative modeling of protein‐protein interactions with pyDock for the new docking challenges

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