In 2016 and 2017, monoclonal antibodies targeting PD-L1, including atezolizumab, durvalumab, and avelumab, were approved by the FDA for the treatment of multiple advanced cancers. And many other ...anti-PD-L1 antibodies are under clinical trials. Recently, the crystal structures of PD-L1 in complex with BMS-936559 and avelumab have been determined, revealing details of the antigen-antibody interactions. However, it is still unknown how atezolizumab and durvalumab specifically recognize PD-L1, although this is important for investigating novel binding sites on PD-L1 targeted by other therapeutic antibodies for the design and improvement of anti-PD-L1 agents. Here, we report the crystal structures of PD-L1 in complex with atezolizumab and durvalumab to elucidate the precise epitopes involved and the structural basis for PD-1/PD-L1 blockade by these antibodies. A comprehensive comparison of PD-L1 interactions with anti-PD-L1 antibodies provides a better understanding of the mechanism of PD-L1 blockade as well as new insights into the rational design of improved anti-PD-L1 therapeutics.
Cancer cells express tumour-specific antigens derived via genetic and epigenetic alterations, which may be targeted by T-cell-mediated immune responses. However, cancer cells can avoid immune ...surveillance by suppressing immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. In recent years, the blockade of checkpoint molecules such as PD-1, PD-L1 and CTLA-4, with monoclonal antibodies has enabled the development of breakthrough therapies in oncology, and four therapeutic antibodies targeting these checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Here, we report the crystal structures of checkpoint molecules in complex with the Fab fragments of therapeutic antibodies, including PD-1/pembrolizumab, PD-1/nivolumab, PD-L1/BMS-936559 and CTLA-4/tremelimumab. These complex structures elucidate the precise epitopes of the antibodies and the molecular mechanisms underlying checkpoint blockade, providing useful information for the improvement of monoclonal antibodies capable of attenuating checkpoint signalling for the treatment of cancer.
In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria‐associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs ...to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER‐anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid‐like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51–VAPB interaction is mediated by the FFAT‐like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM‐tethering function.
SYNOPSIS
The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity.
The crystal structure of the TPR domain of PTPIP51 at 1.45 Å resolution revealed the presence of a lipid‐like serpentine electron density.
PTPIP51 has phospholipid (especially phosphatidic acid) binding and transfer functions in vitro.
Mitochondrial cardiolipin levels are affected by PTPIP51.
The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity.
Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for ...anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.
Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and ...backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement.
The oxidation of cysteine sulphydryl in proteins produces sulphenic acid that can form a reversible disulphide bond with another cysteine. The disulphide bond formation often triggers switches in ...protein structure and activity, especially when the distance between the two cysteine sulphur atoms is longer than the resulting disulphide bond distance. As an early example for the reversible disulphide bond-mediated functional switches, the reduced and oxidized forms of the bacterial transcription factor OxyR were characterized by X-ray crystallography. Recently, the Drosophila vision signalling protein, the association of inactivation-no-afterpotential D (INAD) was analysed by structural and functional methods. The two conserved cysteines of INAD were found to cycle between reduced and oxidized states during the light signal processing in Drosophila eyes, which was achieved by conformation dependent modulation of the disulphide bond redox potential. The production of the hypertension control peptide angiotensins was also shown to be controlled by the reversible disulphide bond in the precursor protein angiotensinogen. The crystal structure of the complex of angiotensiongen with its processing enzyme renin elucidated the role of the disulphide bond in stabilizing the precursor-enzyme complex facilitating the production of angiotensins. The increasing importance of the disulphide bond-mediated redox switches in normal and diseased states has implications in the development of novel antioxidant-based therapeutic approaches.
Cesarean scar pregnancy (CSP) is a rare complication that occurs in less than 1% of ectopic pregnancies, and uterine didelphys is one of the rarest uterine forms. We report a successful laparoscopic ...excision and repair of CSP in a woman with uterine didelphys and a double vagina. A 34-year-old gravida one, para one woman with a history of low transverse cesarean section presented to our hospital with a suspected CSP. She was confirmed to have uterine didelphys with a double vagina during an infertility examination 7 years earlier. Magnetic resonance imaging showed a 2.5-cm gestational sac-like cystic lesion in the lower segment of the right uterus at the cesarean scar. We decided to perform a laparoscopic approach after informing the patient of the surgical procedure. The lower segment of the previous cesarean site was excised with monopolar diathermy to minimize bleeding. We identified the gestational sac in the lower segment of the right uterus, which was evacuated using spoon forceps. The myometrium and serosa of the uterus were sutured layer-by-layer using synthetic absorbable sutures. No remnant gestational tissue was visible on follow-up ultrasonography one month after the surgery. This laparoscopic approach to CSP in a woman with uterine didelphys is an effective and safe method of treatment. In women with uterine anomalies, it is important to confirm the exact location of the gestational sac by preoperative imaging for successful surgery.
The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding ...of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives.
For complete details on the use and execution of this protocol, please refer to Kim et al.1
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•Instructions for purification and characterization of YhaJ effector-binding domain•Steps for crystallization of YhaJ with a DNT derivative•A step-by-step guide for structure determination of the YhaJ:DNT derivative complex
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives.
The Escherichia coli OxyR transcription factor senses H2O2 and is activated through the formation of an intramolecular disulfide bond. Here we present the crystal structures of the regulatory domain ...of OxyR in its reduced and oxidized forms, determined at 2.7 Å and 2.3 Å resolutions, respectively. In the reduced form, the two redox-active cysteines are separated by approximately 17 Å. Disulfide bond formation in the oxidized form results in a significant structural change in the regulatory domain. The structural remodeling, which leads to different oligomeric associations, accounts for the redox-dependent switch in OxyR and provides a novel example of protein regulation by “fold editing” through a reversible disulfide bond formation within a folded domain.
Detection of landmines without harming personnel is a global issue. The bacterial transcription factor YhaJ selectively detects metabolites of explosives, and it can be used as a key component of DNT ...biosensors. However, the wild-type YhaJ has a binding affinity that is not sufficient for the detection of trace amounts of explosives leaked from landmines buried in the soil. Here, we report crystal structures of the effector-binding domain of YhaJ in both the apo- and effector-bound forms. A structural comparison of the two forms revealed that the loop above the primary effector-binding site significantly switches its conformation upon effector binding. The primary effector-binding site involves hydrophobic and polar interactions, having specificity to hydroxyl-substituted benzene compounds. The structures explain the mechanism of activity-enhancing mutations and provide information for the rational engineering of YhaJ biosensors for the sensitive detection of explosives.
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•The bacterial transcription factor YhaJ selectively detects metabolites of explosives•Crystal structures of the apo- and effector-binding forms were determined•The effector-entrance loop significantly switches its conformation•The structures can be used for the rational engineering of sensitive biosensors
Biomaterials; Materials property; Materials science
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