ABSTRACT
The Helicase-like Transcription Factor (HLTF) is a member of the SNF2-family of fork remodelers, primarily studied for its capacity to provide DNA Damage Tolerance (DDT) and to induce replication fork reversal (RFR). HLTF is recruited at stalled forks where both its ATPase motor and HIP116 Rad5p N-terminal (HIRAN) domains are necessary for regulating its interaction with DNA. HIRAN bestows specificity to ssDNA 3'-end and imparts branch migration as well as DNA remodeling capabilities facilitating damage repair. Both expression regulation and mutation rate affect HLTF activity. Gene hypermethylation induces loss of HLTF function, in particular in colorectal cancer (CRC), implying a tumour suppressor role. Surprisingly, a correlation between hypermethylation and HLTF mRNA upregulation has also been observed, even within the same cancer type. In many cancers, both complex mutation patterns and the presence of gene Copy Number Variations (CNVs) have been reported. These conditions affect the amount of functional HLTF and question the physiological role of this fork remodeler. This review offers a systematic collection of the presently strewed information regarding HLTF, its structural and functional characteristics, the multiple roles in DDT and the regulation in cancer progression highlighting new research perspectives.
Subject(s)
DNA Replication , Neoplasms , Transcription Factors , Humans , Transcription Factors/metabolism , Transcription Factors/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Methylation , Gene Expression Regulation, Neoplastic , DNA Repair , DNA Damage , Animals , Mutation , DNA Copy Number VariationsABSTRACT
BACKGROUND: Fluorescent proteins (FPs) are pivotal reagents for flow cytometry analysis or fluorescent microscopy. A new generation of immunoreagents (fluobodies/chromobodies) has been developed by fusing recombinant nanobodies to FPs. METHODS: We analyzed the quality of such biomolecules by a combination of gel filtration and SDS-PAGE to identify artefacts due to aggregation or material degradation. RESULTS: In the SDS-PAGE run, unexpected bands corresponding to separate fluobodies were evidenced and characterized as either degradation products or artefacts that systematically resulted in the presence of specific FPs and some experimental conditions. The elimination of N-terminal methionine from FPs did not impair the appearance of FP fragments, whereas the stability and migration characteristics of some FP constructs were strongly affected by heating in loading buffer, which is a step samples undergo before electrophoretic separation. CONCLUSIONS: In this work, we provide explanations for some odd results observed during the quality control of fluobodies and summarize practical suggestions for the choice of the most convenient FPs to fuse to antibody fragments.
Subject(s)
Electrophoresis, Polyacrylamide Gel , Electrophoresis, Polyacrylamide Gel/methods , Single-Domain Antibodies/chemistry , Humans , Chromatography, Gel , Flow Cytometry/standards , Flow Cytometry/methods , Quality ControlABSTRACT
RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'â3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'â3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.
Subject(s)
Methanocaldococcus , Models, Molecular , Methanocaldococcus/enzymology , Crystallography, X-Ray , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Archaeal Proteins/genetics , Exonucleases/metabolism , Exonucleases/chemistry , Protein Conformation , Amino Acid Sequence , Exodeoxyribonucleases/chemistry , Exodeoxyribonucleases/metabolism , Exodeoxyribonucleases/geneticsABSTRACT
BACKGROUND: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. METHODS: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. RESULTS: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. DISCUSSION: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors.
Subject(s)
Peptide Library , Single-Domain Antibodies , Enzyme-Linked Immunosorbent Assay , Single-Domain Antibodies/pharmacology , Complementarity Determining Regions , EpitopesABSTRACT
Here, we present the crystal structure of the synthetic peptide KE1, which contains four K-coil heptads separated in the middle by the QFLMLMF heptad. The structure determination reveals the presence of a canonical parallel three stranded coiled coil. The geometric characteristics of this structure are compared with other coiled coils with the same topology. Furthermore, for this topology, the analysis of the propensity of the single amino acid to occupy a specific position in the heptad sequence is reported. A number of viral proteins use specialized coiled coil tail needles to inject their genetic material into the host cells. The simplicity and regularity of the coiled coil arrangement made it an attractive system for de novo design of key molecules in drug delivery systems, vaccines, and therapeutics.
ABSTRACT
The interaction between avidin and its counterpart biotin is one of central importance in biology and has been reproposed and studied at length. However, the binding pocket of avidin is prone to promiscuous binding, able to accommodate even non-biotinylated ligands. Comprehending the factors that distinguish the extremely strong interaction with biotin to other ligands is an important step to fully picture the thermodynamics of these low-affinity complexes. Here, we present the complex between chicken white egg avidin and theophylline (TEP), the xanthine derivative used in the therapy of asthma. In the crystal structure, TEP lies in the biotin-binding pocket with the same orientation and planarity of the aromatic ring of 8-oxodeoxyguanosine. Indeed, its affinity for avidin measured by isothermal titration calorimetry is in the same µM range as those obtained for the previously characterized nucleoside derivatives. By the use of molecular dynamic simulations, we have investigated the most important intermolecular interactions occurring in the avidin-TEP binding pocket and compared them with those obtained for the avidin 8-oxodeoxyguanosine and avidin-biotin complexes. These results testify the capability of avidin to complex purely aromatic molecules.
Subject(s)
Avidin , Biotin , Avidin/chemistry , Avidin/metabolism , Biotin/chemistry , Biotin/metabolism , Theophylline , Ligands , ThermodynamicsABSTRACT
Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.
Subject(s)
Body Fluids , Extracellular Vesicles , Single-Domain Antibodies , Biomarkers/metabolism , Body Fluids/metabolism , Extracellular Vesicles/metabolism , Proteins/metabolism , Single-Domain Antibodies/metabolism , Tetraspanin 30ABSTRACT
Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO2 and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Viral , Enzyme-Linked Immunosorbent Assay , Humans , Protein Binding , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistryABSTRACT
Members of the tripartite motif family of E3 ubiquitin ligases are characterized by the presence of a conserved N-terminal module composed of a RING domain followed by one or two B-box domains, a coiled-coil and a variable C-terminal region. The RING and B-box are both Zn-binding domains but, while the RING is found in a large number of proteins, the B-box is exclusive to the tripartite motif (TRIM) family members in metazoans. Whereas the RING has been extensively characterized and shown to possess intrinsic E3 ligase catalytic activity, much less is known about the role of the B-box domains. In this study, we adopted an in vitro approach using recombinant point- and deletion-mutants to characterize the contribution of the TRIM32 Zn-binding domains to the activity of this E3 ligase that is altered in a genetic form of muscular dystrophy. We found that the RING domain is crucial for E3 ligase activity and E2 specificity, whereas a complete B-box domain is involved in chain assembly rate modulation. Further, in vitro, the RING domain is necessary to modulate TRIM32 oligomerization, whereas, in cells, both the RING and B-box cooperate to specify TRIM32 subcellular localization, which if altered may impact the pathogenesis of diseases.
Subject(s)
Muscular Dystrophies, Limb-Girdle/genetics , Mutation/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Tripartite Motif Proteins/chemistry , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Zinc/metabolism , Animals , Biocatalysis , Cell Line , Humans , Mice , Mutant Proteins/metabolism , Protein Domains , Protein Multimerization , Transcription Factors/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
p15PAF is an oncogenic intrinsically disordered protein that regulates DNA replication and lesion bypass by interacting with the human sliding clamp PCNA. In the absence of DNA, p15PAF traverses the PCNA ring via an extended PIP-box that contacts the sliding surface. Here, we probed the atomic-scale structure of p15PAF-PCNA-DNA ternary complexes. Crystallography and MD simulations show that, when p15PAF occupies two subunits of the PCNA homotrimer, DNA within the ring channel binds the unoccupied subunit. The structure of PCNA-bound p15PAF in the absence and presence of DNA is invariant, and solution NMR confirms that DNA does not displace p15PAF from the ring wall. Thus, p15PAF reduces the available sliding surfaces of PCNA, and may function as a belt that fastens the DNA to the clamp during synthesis by the replicative polymerase (pol δ). This constraint, however, may need to be released for efficient DNA lesion bypass by the translesion synthesis polymerase (pol η). Accordingly, our biochemical data show that p15PAF impairs primer synthesis by pol η-PCNA holoenzyme against both damaged and normal DNA templates. In light of our findings, we discuss the possible mechanistic roles of p15PAF in DNA replication and suppression of DNA lesion bypass.
Subject(s)
Carrier Proteins/chemistry , DNA/chemistry , Intrinsically Disordered Proteins/chemistry , Proliferating Cell Nuclear Antigen/chemistry , Carrier Proteins/genetics , Crystallography, X-Ray , DNA/genetics , DNA Polymerase III/chemistry , DNA Polymerase III/genetics , DNA Replication/genetics , DNA-Binding Proteins , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Escherichia coli/genetics , Holoenzymes/chemistry , Holoenzymes/genetics , Humans , Intrinsically Disordered Proteins/genetics , Magnetic Resonance Spectroscopy , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Proliferating Cell Nuclear Antigen/geneticsABSTRACT
Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+ channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA). We combined electrophysiology and molecular dynamics simulations to examine how DMA interacts with the pore and permeates through it. Due to the presence of hydrophobic groups, DMA enters easily in the channel and, unlike the alkali cations, does not need to cross any barrier. We also show that while the crystal structure is consistent with the presence of a single DMA ion at full occupancy, the channel is able to conduct a sizable current of DMA ions only when two ions are present inside the channel. Moreover, the second DMA ion dramatically changes the free energy landscape, destabilizing the crystallographic binding site and lowering by almost 25 kJ/mol the binding affinity between DMA and the channel. Based on the results of the simulation the experimental electron density maps can be re-interpreted with the presence of a second ion at lower occupancy. In this mechanism the flexibility of the channel plays a key role, extending the classical multi-ion permeation paradigm in which conductance is enhanced by the plain interaction between the ions.
Subject(s)
Cyclic Nucleotide-Gated Cation Channels/metabolism , Organic Cation Transport Proteins/physiology , Animals , Biophysical Phenomena , Cations/metabolism , Computer Simulation , Cyclic Nucleotide-Gated Cation Channels/physiology , Dimethylamines/metabolism , Gap Junctions/metabolism , Membrane Potentials/physiology , Molecular Dynamics Simulation , Oocytes/physiology , Quaternary Ammonium Compounds/metabolism , Sodium/metabolism , Xenopus laevisABSTRACT
The proliferating cell nuclear antigen (PCNA) sliding clamp lies at the heart of the accurate duplication of eukaryotic genomes. While the outer surface of the PCNA ring interacts with polymerases and other factors, the role of the inner wall facing the DNA is elusive. Recent evidence shows that conserved basic residues in the PCNA central channel create a specific surface that recognizes the DNA backbone and enables the clamp to slide by rotationally tracking the DNA helix. The sliding surface can be modulated (i) through lysine acetylation, which triggers PCNA degradation during nucleotide excision repair (NER) and stimulates repair by homologous recombination (HR) or (ii) through binding of the protein factor p15PAF, which turns off DNA lesion bypass. Thus, the inner surface of PCNA is unexpectedly highly regulated to control resistance to DNA damage. From a structural viewpoint, we reflect on these findings that open a new perspective on PCNA function and offer opportunities to develop tools to manipulate the DNA damage response in cancer treatment.
Subject(s)
DNA/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Animals , DNA/genetics , DNA Damage , DNA Replication , Humans , Models, Molecular , Neoplasms/genetics , Neoplasms/metabolism , Proliferating Cell Nuclear Antigen/chemistry , Protein ConformationABSTRACT
Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a 'cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA-DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.
Subject(s)
DNA/chemistry , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Crystallography, X-Ray , DNA Polymerase III/chemistry , DNA Polymerase III/metabolism , DNA Replication , DNA, B-Form/chemistry , DNA, B-Form/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Dynamics Simulation , Mutation , Proliferating Cell Nuclear Antigen/geneticsABSTRACT
GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.
Subject(s)
Chromosomal Proteins, Non-Histone/chemistry , DNA-Binding Proteins/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Humans , Microscopy, Electron , Models, Molecular , Protein Multimerization , Scattering, Small AngleABSTRACT
RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix ß-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.
Subject(s)
DNA/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , RecQ Helicases/chemistry , RecQ Helicases/metabolism , Adenosine Triphosphatases/metabolism , Computational Biology , Crystallography, X-Ray , DNA/chemistry , DNA/genetics , Databases, Protein , Humans , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutation/genetics , Protein Binding , Protein Domains , Protein Structure, Tertiary , RecQ Helicases/geneticsABSTRACT
Using our previously reported maps of the electrostatic surface of horse heart ferri- and ferro-cyt c, comparisons were made between the complementary electrostatic surfaces of three cyt c peroxidase-cyt c complexes and the photosynthetic reaction center-cyt c complex, considering both iron oxidation states. The results obtained were consistent with a sliding mechanism for the electron shuttle on the surface of the protein complexes, promoted by the change in iron oxidation state. This mechanism was found to be in agreement with theoretical and NMR studies reported in the literature. Importantly, the analysis also provided a rationale for recognition of nonproductive associations. As we have previously reported the same conclusion on examination of redox partners of cyt c in the mitochondrial respiratory pathway, our hypothesis is that the proposed mechanism could represent a general exit strategy of monoheme cyts c and c2 in electron transfer complexes.
Subject(s)
Cell Respiration/physiology , Cytochromes c/chemistry , Cytochromes c/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Animals , Bacteria/metabolism , Electron Transport , Horses , Kinetics , Models, Molecular , Oxidation-Reduction , Peroxidase , Protein Conformation , Saccharomyces cerevisiae/metabolismABSTRACT
Cyclic nucleotide-gated (CNG) ion channels, despite a significant homology with the highly selective K(+) channels, do not discriminate among monovalent alkali cations and are permeable also to several organic cations. We combined electrophysiology, molecular dynamics (MD) simulations, and X-ray crystallography to demonstrate that the pore of CNG channels is highly flexible. When a CNG mimic is crystallized in the presence of a variety of monovalent cations, including Na(+), Cs(+), and dimethylammonium (DMA(+)), the side chain of Glu66 in the selectivity filter shows multiple conformations and the diameter of the pore changes significantly. MD simulations indicate that Glu66 and the prolines in the outer vestibule undergo large fluctuations, which are modulated by the ionic species and the voltage. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels.
Subject(s)
Cyclic Nucleotide-Gated Cation Channels/chemistry , Cyclic Nucleotide-Gated Cation Channels/metabolism , Ion Channel Gating/physiology , Protein Conformation , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cations, Monovalent/metabolism , Cattle , Cesium/metabolism , Crystallography, X-Ray , Cyclic Nucleotide-Gated Cation Channels/genetics , Female , Ion Channel Gating/genetics , Ion Transport/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Molecular Dynamics Simulation , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation, Missense , Oocytes/metabolism , Oocytes/physiology , Patch-Clamp Techniques , Sequence Homology, Amino Acid , Sodium/metabolism , Xenopus laevisABSTRACT
Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (Rfact of 13.8% and Rfree of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds.
Subject(s)
Bacterial Proteins/chemistry , Oxidoreductases/chemistry , Shewanella/chemistry , Binding Sites , Crystallography, X-Ray , Heme/chemistry , Hydrogen Bonding , Models, Molecular , Protein Structure, TertiaryABSTRACT
A new pseudopeptide epoxide inhibitor, designed for irreversible binding to HIV protease (HIV-PR), has been synthesized and characterized in solution and in the solid state. However, the crystal structure of the complex obtained by inhibitor-enzyme cocrystallization revealed that a minor isomer, with inverted configuration of the epoxide carbons, has been selected by HIV-PR during crystallization. The structural characterization of the well-ordered pseudopeptide, inserted in the catalytic channel with its epoxide group intact, provides deeper insights into inhibitor binding and HIV-PR stereoselectivity, which aids development of future epoxide-based HIV inhibitors.