Erik Zavala, Stephen Dansereau, Michael J Burke, James M Lipchock, Federica Maschietto, Victor Batista, J Patrick Loria
PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.
PHPT1 是一种组氨酸磷酸酶,通过其催化活性调节真核生物的信号转导。在这里,我们结合溶液核磁共振、分子动力学和生化实验,对 PHPT1 的结构和动力学进行了分析。我们确定了 R78 胍基与 C 端 Y125 上的羧基之间形成的盐桥,它对配体的结合至关重要。通过在 C 端添加一个甘氨酸残基(G126)来破坏该盐桥,会导致催化活性降低,与伪底物对硝基苯磷酸(pNPP)以及活性位点抑制剂苯膦酸(PPA)的结合亲和力下降。我们通过核磁共振化学位移、15N 驰豫测量和分子动力学轨迹分析表明,移除这种盐桥会导致活性位点的结构和动力学发生改变,从而对酶的功能产生重大影响,并证实了这种静电相互作用的重要性。
{"title":"A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.","authors":"Erik Zavala, Stephen Dansereau, Michael J Burke, James M Lipchock, Federica Maschietto, Victor Batista, J Patrick Loria","doi":"10.1002/pro.5009","DOIUrl":"10.1002/pro.5009","url":null,"abstract":"<p><p>PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, <sup>15</sup>N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11094782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140922962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jorge Luis Medrano-Cerano, Luis Fernando Cofas-Vargas, Eduardo Leyva, Jesús Antonio Rauda-Ceja, Mateo Calderón-Vargas, Patricia Cano-Sánchez, Gustavo Titaux-Delgado, Carolina Monserrath Melchor-Meneses, Andrés Hernández-Arana, Federico Del Río-Portilla, Enrique García-Hernández
Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.
{"title":"Decoding the mechanism governing the structural stability of wheat germ agglutinin and its isolated domains: A combined calorimetric, NMR, and MD simulation study.","authors":"Jorge Luis Medrano-Cerano, Luis Fernando Cofas-Vargas, Eduardo Leyva, Jesús Antonio Rauda-Ceja, Mateo Calderón-Vargas, Patricia Cano-Sánchez, Gustavo Titaux-Delgado, Carolina Monserrath Melchor-Meneses, Andrés Hernández-Arana, Federico Del Río-Portilla, Enrique García-Hernández","doi":"10.1002/pro.5020","DOIUrl":"10.1002/pro.5020","url":null,"abstract":"<p><p>Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11094770/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140922991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Many small globular proteins exist in only two states-the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability. We focus on one such interaction, that between amide and aromatic groups. We provide a statistically convincing case for quantitative, linear entropy-enthalpy compensation in forming aromatic-amide interactions using published model compound transfer-free energy data.
{"title":"Quantitative entropy-enthalpy compensation in intraprotein interactions from model compound data.","authors":"Thomas W Redvanly, Gary J Pielak","doi":"10.1002/pro.5013","DOIUrl":"10.1002/pro.5013","url":null,"abstract":"<p><p>Many small globular proteins exist in only two states-the physiologically relevant folded state and an inactive unfolded state. The active state is stabilized by numerous weak attractive contacts, including hydrogen bonds, other polar interactions, and the hydrophobic effect. Knowledge of these interactions is key to understanding the fundamental equilibrium thermodynamics of protein folding and stability. We focus on one such interaction, that between amide and aromatic groups. We provide a statistically convincing case for quantitative, linear entropy-enthalpy compensation in forming aromatic-amide interactions using published model compound transfer-free energy data.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11135021/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141162465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.
{"title":"Insights on the G protein-coupled receptor helix 8 solution structure and orientation using a neurotensin receptor 1 peptide.","authors":"James B Bower, Scott A Robson, Joshua J Ziarek","doi":"10.1002/pro.4976","DOIUrl":"10.1002/pro.4976","url":null,"abstract":"<p><p>G-protein coupled receptors (GPCRs) are the largest class of membrane proteins encoded in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminal tail (Ctail). Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the Ctail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and Ctail (H8-Ctail) to investigate its structural stability, conformational dynamics, and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift-derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement NMR. Taken together, our studies reveal the H8-Ctail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11099793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140959223","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shankar Raj Devkota, Pramod Aryal, Matthew C J Wilce, Richard J Payne, Martin J Stone, Ram Prasad Bhusal
Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.
蜱虫会在唾液中产生趋化因子结合蛋白,即 Evasins,以破坏宿主的免疫反应。evasins 与趋化因子结合,从而抑制其同源趋化因子受体的活化,从而抑制白细胞的募集和炎症反应。我们最近描述了 A3 亚类 evasins,与其他 A 类 evasins 一样,它们只针对 CC 趋化因子,但似乎使用不同的结合位点结构来控制 CC 趋化因子的目标选择性。我们现在描述 A3 类 evasin EVA-ACA1001 识别趋化因子的结构基础。EVA-ACA1001 几乎能与所有人类 CC 趋化因子结合,并抑制受体活化。EVA-ACA1001 的截短突变体表明,与 A1 类 evasins 不同,EVA-ACA1001 的 N 端和 C 端在趋化因子结合中的作用微乎其微。为了了解其广泛识别趋化因子的结构基础,我们测定了 EVA-ACA1001 与人类趋化因子 CCL16 复合物的晶体结构。EVA-ACA1001与CCL16的CC基团形成骨干-骨干相互作用,这是所有A类evasin-趋化因子复合物的保守特征。EVA-ACA1001 中的疏水口袋由几个芳香族侧链和 A3 类 evasins 独有的二硫键形成,可容纳紧随 CCL16 的 CC 基序之后的残基("CC + 1 残基")。这种相互作用与 EVA-AAM1001 共享,而 EVA-AAM1001 是迄今为止唯一具有特征的其他 A3 类 evasins,这表明它可能是 A3 类 evasins 广泛识别 CC 趋化因子的共同机制。
{"title":"Structural basis of chemokine recognition by the class A3 tick evasin EVA-ACA1001.","authors":"Shankar Raj Devkota, Pramod Aryal, Matthew C J Wilce, Richard J Payne, Martin J Stone, Ram Prasad Bhusal","doi":"10.1002/pro.4999","DOIUrl":"10.1002/pro.4999","url":null,"abstract":"<p><p>Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the \"CC + 1 residue\") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11081419/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140899359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.
{"title":"Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA.","authors":"Risa Asano, Miyu Takeuchi, Makoto Nakakido, Sho Ito, Chihiro Aikawa, Takeshi Yokoyama, Akinobu Senoo, Go Ueno, Satoru Nagatoishi, Yoshikazu Tanaka, Ichiro Nakagawa, Kouhei Tsumoto","doi":"10.1002/pro.5017","DOIUrl":"10.1002/pro.5017","url":null,"abstract":"<p><p>Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.</p>","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11094767/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140922920","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Serum amyloid A (SAA) is a highly conserved acute‐phase protein that plays roles in activating multiple pro‐inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high‐density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein–protein interaction sites on SAA. While high‐resolution structures for lipid‐free, or apo‐, forms of SAA have been reported, their relationship with the HDL‐bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC‐MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid‐free and lipid‐bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring‐like structures reported for lipid‐free ApoA‐I. These SAA octamers represent a previously uncharacterized structure for lipid‐free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA‐I. The SAA–lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid‐free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid‐containing particles such as HDL. This study suggests a new model for SAA–lipid interactions and provides new insight into how SAA might stabilize protein‐lipid nanodiscs or even replace ApoA‐I as a scaffold for HDL particles during inflammation.
血清淀粉样蛋白 A(SAA)是一种高度保守的急性期蛋白,在急性炎症反应期间可激活多种促炎症途径,通常被用作炎症的生物标志物。它通过改善损伤部位脂质和胆固醇的清除,在组织修复中发挥有益作用。在慢性炎症性疾病患者中,SAA 水平的升高可能会加重潜在疾病的严重程度。循环中的 SAA 大部分与脂蛋白结合,主要是高密度脂蛋白(HDL)。与 HDL 的相互作用不仅能稳定 SAA,还能改变其功能特性,这可能是通过改变 SAA 上蛋白质与蛋白质相互作用位点的可及性实现的。虽然无脂或脂蛋白形式 SAA 的高分辨率结构已有报道,但它们与高密度脂蛋白结合形式的蛋白质以及 SAA 与脂质结合的其他可能机制之间的关系尚未确定。在这里,我们使用了多种生物物理技术,包括 SAXS、TEM、SEC-MALS、原生凝胶电泳、戊二醛交联和胰蛋白酶消化,来描述 SAA 的无脂和脂质结合形式。SAXS和TEM数据显示,SAA存在可溶性八聚体,其结构与已报道的无脂载脂蛋白A-I的环状结构相似。这些 SAA 八聚体代表了无脂 SAA 以前未曾表征的结构,能够构建与载脂蛋白 ApoA-I 形成的形态相似的脂质纳米盘。SAA-脂质纳米圆片含有四个 SAA 分子,其外部尺寸与无脂 SAA 八聚体相似,这表明 SAA 与 HDL 等含脂微粒的相互作用可能只需要相对较少的构象重排。这项研究为 SAA 与脂质的相互作用提出了一个新的模型,并为了解 SAA 如何在炎症期间稳定蛋白-脂质纳米盘,甚至取代载脂蛋白 ApoA-I 成为 HDL 颗粒的支架提供了新的见解。
{"title":"Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs","authors":"Asal Nady, Sean E. Reichheld, Simon Sharpe","doi":"10.1002/pro.4983","DOIUrl":"https://doi.org/10.1002/pro.4983","url":null,"abstract":"Serum amyloid A (SAA) is a highly conserved acute‐phase protein that plays roles in activating multiple pro‐inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high‐density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein–protein interaction sites on SAA. While high‐resolution structures for lipid‐free, or apo‐, forms of SAA have been reported, their relationship with the HDL‐bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC‐MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid‐free and lipid‐bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring‐like structures reported for lipid‐free ApoA‐I. These SAA octamers represent a previously uncharacterized structure for lipid‐free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA‐I. The SAA–lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid‐free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid‐containing particles such as HDL. This study suggests a new model for SAA–lipid interactions and provides new insight into how SAA might stabilize protein‐lipid nanodiscs or even replace ApoA‐I as a scaffold for HDL particles during inflammation.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140801597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Itzell Hernández‐Sánchez, Tobias Rindfleisch, Jessica Alpers, Martin Dulle, Christopher J. Garvey, Patrick Knox‐Brown, Markus S. Miettinen, Gergely Nagy, Julio M. Pusterla, Agata Rekas, Keyun Shou, Andreas M. Stadler, Dirk Walther, Martin Wolff, Ellen Zuther, Anja Thalhammer
Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration‐sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra‐ and inter‐monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α‐helical molecular recognition feature (α‐MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right‐handed coiled‐coils might be a recurring theme for homo‐ and hetero‐oligomerization of LEA proteins.
{"title":"Functional in vitro diversity of an intrinsically disordered plant protein during freeze–thawing is encoded by its structural plasticity","authors":"Itzell Hernández‐Sánchez, Tobias Rindfleisch, Jessica Alpers, Martin Dulle, Christopher J. Garvey, Patrick Knox‐Brown, Markus S. Miettinen, Gergely Nagy, Julio M. Pusterla, Agata Rekas, Keyun Shou, Andreas M. Stadler, Dirk Walther, Martin Wolff, Ellen Zuther, Anja Thalhammer","doi":"10.1002/pro.4989","DOIUrl":"https://doi.org/10.1002/pro.4989","url":null,"abstract":"Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration‐sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the <jats:italic>Arabidopsis thaliana</jats:italic> LEA protein COR15A found <jats:italic>in vitro</jats:italic> is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra‐ and inter‐monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α‐helical molecular recognition feature (α‐MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right‐handed coiled‐coils might be a recurring theme for homo‐ and hetero‐oligomerization of LEA proteins.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140801688","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Billy K. Poon, Thomas C. Terwilliger, Paul D. Adams
Advances in machine learning have enabled sufficiently accurate predictions of protein structure to be used in macromolecular structure determination with crystallography and cryo‐electron microscopy data. The Phenix software suite has AlphaFold predictions integrated into an automated pipeline that can start with an amino acid sequence and data, and automatically perform model‐building and refinement to return a protein model fitted into the data. Due to the steep technical requirements of running AlphaFold efficiently, we have implemented a Phenix‐AlphaFold webservice that enables all Phenix users to run AlphaFold predictions remotely from the Phenix GUI starting with the official 1.21 release. This webservice will be improved based on how it is used by the research community and the future research directions for Phenix.
{"title":"The Phenix‐AlphaFold webservice: Enabling AlphaFold predictions for use in Phenix","authors":"Billy K. Poon, Thomas C. Terwilliger, Paul D. Adams","doi":"10.1002/pro.4992","DOIUrl":"https://doi.org/10.1002/pro.4992","url":null,"abstract":"Advances in machine learning have enabled sufficiently accurate predictions of protein structure to be used in macromolecular structure determination with crystallography and cryo‐electron microscopy data. The Phenix software suite has AlphaFold predictions integrated into an automated pipeline that can start with an amino acid sequence and data, and automatically perform model‐building and refinement to return a protein model fitted into the data. Due to the steep technical requirements of running AlphaFold efficiently, we have implemented a Phenix‐AlphaFold webservice that enables all Phenix users to run AlphaFold predictions remotely from the Phenix GUI starting with the official 1.21 release. This webservice will be improved based on how it is used by the research community and the future research directions for Phenix.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140634469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mikhail Baloban, Ludmila A. Kasatkina, Vladislav V. Verkhusha
Optogenetic tools (OTs) operating in the far‐red and near‐infrared (NIR) region offer advantages for light‐controlling biological processes in deep tissues and spectral multiplexing with fluorescent probes and OTs acting in the visible range. However, many NIR OTs suffer from background activation in darkness. Through shortening linkers, we engineered a novel NIR OT, iLight2, which exhibits a significantly reduced background activity in darkness, thereby increasing the light‐to‐dark activation contrast. The resultant optimal configuration of iLight2 components suggests a molecular mechanism of iLight2 action. Using a biliverdin reductase knock‐out mouse model, we show that iLight2 exhibits advanced performance in mouse primary cells and deep tissues in vivo. Efficient light‐controlled cell migration in wound healing cellular model demonstrates the possibility of using iLight2 in therapy and, overall, positions it as a valuable addition to the NIR OT toolkit for gene transcription applications.
在远红外和近红外(NIR)区域工作的光遗传工具(OTs)在深部组织的光控生物过程以及与荧光探针和在可见光范围工作的 OTs 进行光谱复用方面具有优势。然而,许多近红外 OT 在黑暗中会受到背景激活的影响。通过缩短连接体,我们设计出一种新型近红外 OT iLight2,它在黑暗中的背景活性显著降低,从而提高了明暗激活对比度。iLight2 成分的最佳配置结果表明了 iLight2 的分子作用机制。利用胆绿素还原酶基因敲除小鼠模型,我们证明了 iLight2 在小鼠原代细胞和体内深层组织中表现出的先进性能。伤口愈合细胞模型中高效的光控细胞迁移证明了 iLight2 用于治疗的可能性,总体而言,iLight2 是近红外 OT 工具包中基因转录应用的重要补充。
{"title":"iLight2: A near‐infrared optogenetic tool for gene transcription with low background activation","authors":"Mikhail Baloban, Ludmila A. Kasatkina, Vladislav V. Verkhusha","doi":"10.1002/pro.4993","DOIUrl":"https://doi.org/10.1002/pro.4993","url":null,"abstract":"Optogenetic tools (OTs) operating in the far‐red and near‐infrared (NIR) region offer advantages for light‐controlling biological processes in deep tissues and spectral multiplexing with fluorescent probes and OTs acting in the visible range. However, many NIR OTs suffer from background activation in darkness. Through shortening linkers, we engineered a novel NIR OT, iLight2, which exhibits a significantly reduced background activity in darkness, thereby increasing the light‐to‐dark activation contrast. The resultant optimal configuration of iLight2 components suggests a molecular mechanism of iLight2 action. Using a biliverdin reductase knock‐out mouse model, we show that iLight2 exhibits advanced performance in mouse primary cells and deep tissues <jats:italic>in vivo</jats:italic>. Efficient light‐controlled cell migration in wound healing cellular model demonstrates the possibility of using iLight2 in therapy and, overall, positions it as a valuable addition to the NIR OT toolkit for gene transcription applications.","PeriodicalId":20761,"journal":{"name":"Protein Science","volume":null,"pages":null},"PeriodicalIF":8.0,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140634349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}