8 research outputs found
Specificity of iron-phytosiderophore transporter
Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter of Fe(III)-phytosiderophores in barley that is responsible for iron acquisition from the soil. In contrast, maize Zea mays, yellow stripe 1 (ZmYS1) possesses broad substrate specificity. In this study, a quantitative evaluation of the transport activities of HvYS1 and ZmYS1 chimera proteins revealed that the seventh extracellular membrane loop is essential for substrate specificity. The loop peptides of both transporters were prepared and analysed by circular dichroism and NMR. The spectra revealed a higher propensity for α-helical conformation of the HvYS1 loop peptide and a largely disordered structure for that of ZmYS1. These structural differences are potentially responsible for the substrate specificities of the transporters
Distal Regulation of Heme Binding of Heme Oxygenase‑1 Mediated by Conformational Fluctuations
Heme
oxygenase-1 (HO-1) is an enzyme that catalyzes the oxidative
degradation of heme. Since free heme is toxic to cells, rapid degradation
of heme is important for maintaining cellular health. There have been
useful mechanistic studies of the HO reaction based on crystal structures;
however, how HO-1 recognizes heme is not completely understood because
the crystal structure of heme-free rat HO-1 lacks electron densities
for A-helix that ligates heme. In this study, we characterized conformational
dynamics of HO-1 using NMR to elucidate the mechanism by which HO-1
recognizes heme. NMR relaxation experiments showed that the heme-binding
site in heme-free HO-1 fluctuates in concert with a surface-exposed
loop and transiently forms a partially unfolded structure. Because
the fluctuating loop is located over 17 Å distal from the heme-binding
site and its conformation is nearly identical among different crystal
structures including catalytic intermediate states, the function of
the loop has been unexamined. In the course of elucidating its function,
we found interesting mutations in this loop that altered activity
but caused little change to the conformation. The Phe79Ala mutation
in the loop changed the conformational dynamics of the heme-binding
site. Furthermore, the heme binding kinetics of the mutant was slower
than that of the wild type. Hence, we concluded that the distal loop
is involved in the regulation of the conformational change for heme
binding through the conformational fluctuations. Similar to other
enzymes, HO-1 effectively promotes its function using the identified
distal sites, which might be potential targets for protein engineering
Solid-State NMR Spectra of Lipid-Anchored Proteins under Magic Angle Spinning
Solid-state NMR is a promising tool
for elucidating membrane-related
biological phenomena. We achieved the measurement of high-resolution
solid-state NMR spectra for a lipid-anchored protein embedded in lipid
bilayers under magic angle spinning (MAS). To date, solid-state NMR
measurements of lipid-anchored proteins have not been accomplished
due to the difficulty in supplying sufficient amount of stable isotope
labeled samples in the overexpression of lipid-anchored proteins requiring
complex posttranslational modification. We designed a pseudo lipid-anchored
protein in which the protein component was expressed in E. coli and attached to a chemically synthesized
lipid-anchor mimic. Using two types of membranes, liposomes and bicelles,
we demonstrated different types of insertion procedures for lipid-anchored
protein into membranes. In the liposome sample, we were able to observe
the cross-polarization and the <sup>13</sup>C–<sup>13</sup>C chemical shift correlation spectra under MAS, indicating that the
liposome sample can be used to analyze molecular interactions using
dipolar-based NMR experiments. In contrast, the bicelle sample showed
sufficient quality of spectra through scalar-based experiments. The
relaxation times and protein–membrane interaction were capable
of being analyzed in the bicelle sample. These results demonstrated
the applicability of two types of sample system to elucidate the roles
of lipid-anchors in regulating diverse biological phenomena
Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance
Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer
Glycyrrhizin Derivatives Suppress Cancer Chemoresistance by Inhibiting Progesterone Receptor Membrane Component 1
Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in various cancer cells and contributes to tumor progression. We have previously shown that PGRMC1 forms a unique heme-stacking functional dimer to enhance EGF receptor (EGFR) activity required for cancer proliferation and chemoresistance, and the dimer dissociates by carbon monoxide to attenuate its biological actions. Here, we determined that glycyrrhizin (GL), which is conventionally used to ameliorate inflammation, specifically binds to heme-dimerized PGRMC1. Binding analyses using isothermal titration calorimetry revealed that some GL derivatives, including its glucoside-derivative (GlucoGL), bind to PGRMC1 potently, whereas its aglycone, glycyrrhetinic acid (GA), does not bind. GL and GlucoGL inhibit the interaction between PGRMC1 and EGFR, thereby suppressing EGFR-mediated signaling required for cancer progression. GL and GlucoGL significantly enhanced EGFR inhibitor erlotinib- or cisplatin (CDDP)-induced cell death in human colon cancer HCT116 cells. In addition, GL derivatives suppressed the intracellular uptake of low-density lipoprotein (LDL) by inhibiting the interaction between PGRMC1 and the LDL receptor (LDLR). Effects on other pathways cannot be excluded. Treatment with GlucoGL and CDDP significantly suppressed tumor growth following xenograft transplantation in mice. Collectively, this study indicates that GL derivatives are novel inhibitors of PGRMC1 that suppress cancer progression, and our findings provide new insights for cancer treatment