Conformational dynamics of the anthrax lethal factor catalytic center

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn(2+) cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region.     

Structure-function discrimination of the N- and C- catalytic domains of human angiotensin-converting enzyme: implications for Cl- activation and peptide hydrolysis mechanisms

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two sequence homologous protein domains (ACE_N and ACE_C) possessing several biochemical features that differentiate the two active sites (i.e. chloride ion activation). Based on the recently solved X-ray structure of testis angiotensin-converting enzyme (tACE), the 3D structure of ACE_N was modeled. Electrostatic potential calculations reveal that the ACE_N binding groove is significantly more positively charged than the ACE_C, which provides a first rationalization for their functional discrimination. The chloride ion pore for Cl2 (one of the two chloride ions revealed in the X-ray structure of tACE) that connects the external solution with the inner part of the protein was identified on the basis of an extended network of water molecules. Comparison of ACE_C with the X-ray structure of the prokaryotic ClC Cl(-) channel from Salmonella enterica serovar typhimurium demonstrates a common molecular basis of anion selectivity. The critical role for Cl2 as an ionic switch is emphasized. Sequence and structural comparison between ACE_N and ACE_C and of other proteins of the gluzincin family highlights key residues that could be responsible for the peptide hydrolysis mechanism. Currently available mutational and substrate hydrolysis data for both domains are evaluated and are consistent with the predicted model.     

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c. The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions. The process can be described in terms of protein fragments behaving as unfolding units of different stability. The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility.     

Solution structure of reduced horse heart cytochrome c

 In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined. The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination. Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme. Also some hydrogen bonds are changed upon reduction of the heme iron. A substantial similarity is observed for the backbone fold, independently of the oxidation state. At variance, some meaningful differences are observed in the orientation of a few side chains. These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae. The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein. We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. Received: 8 June 1998 / Accepted: 13 October 1998     

Insights into the anthrax lethal factor–substrate interaction and selectivity using docking and molecular dynamics simulations

The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn‐dependent, highly specific metalloprotease with a molecular mass of ～90 kDa that cleaves most isoforms of the family of mitogen‐activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK‐based synthetic peptide provided structure‐activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF‐MEK/MKK interaction along the catalytic channel up to a distance of 20 Å from the zinc atom. This residue‐specific view of the enzyme‐substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot‐spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction.     

NMR structural elucidation of myelin basic protein epitope 83–99 implicated in multiple sclerosis

Myelin basic protein peptide 83–99 (MBP83–99) is the most immunodominant epitope playing a significant role in the multiple sclerosis (MS), an autoimmune disease of the central nervous system. Many peptide analogues, linear or cyclic have been designed and synthesized based on this segment in order to inhibit the experimental autoimmune encephalomyelitis, the best well-known animal model of MS. In this study, the solution structural motif of MBP_83–99 has been performed using 2D ¹H-NMR spectroscopy in dimethyl sulfoxide. A rather extended conformation, along with the formation of a well defined α-helix spanning residues Val⁸⁷–Phe⁹⁰ is proposed, as no long-range NOE are presented. Moreover, the residues of MBP peptide that are important for T-cell receptor recognition are solvent exposed. The spatial arrangement of the side chain all over the sequence of our NMR based model exhibits great similarity with the solid state model, while both TCR contacts occupy the same region in space.     

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (M^pro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective M^pro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative M^pro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as M^pro inhibitors. In this manner, we aimed to complement enzymatic activity assays of M^pro performed by other groups with information on ligand affinity. We have made the M^pro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of M^pro STD-NMR data, thereby accelerating ongoing drug design efforts.

     

Mechanism of Cu(A) assembly

Copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. Here we show that a new periplasmic protein (PCu(A)C) selectively inserts Cu(I) ions into subunit II of Thermus thermophilus ba(3) oxidase to generate a native Cu(A) site. The purported metallochaperone Sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the Cu(A) cysteine ligands.     

Zinc binding in peptide models of angiotensin-I converting enzyme active sites studied through 1H-NMR and chemical shift perturbation mapping

We report the design and synthesis through solid phase 9-flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin-I converting enzyme active sites possessing the general sequence HEMGHX(23)EAIGDX(3). Their zinc-binding properties were monitored in solution through high-resolution (1)H-NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme.     

Synthesis and Biological Evaluation of New GnRH Analogues on Pituitary and Breast Cancer Cells

GnRH analogues have been extensively used in oncology to induce reversible chemical castration due to their hypophysiotropic action. In addition to that, it has recently been shown that many malignant cells, such as breast cancer cells, locally produce GnRH and express the GnRH receptor/s. In order to investigate the structure-activity relationships in both pituitary and extrapituitary biological systems, we synthesized eight new GnRH analogues with modifications in the N-terminal part and/or in position 6 and studied their pituitary binding affinity (in αT3-1 cell membranes) and effect on breast cancer (MCF-7) cell proliferation. 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC) was incorporated instead of pGlu¹-His²- and/or Gly⁶ was substituted by α-aminoisobutyric acid, D-Leu and D-Lys (alone or covalently linked to Gly, Ala, Sar, ATPC). Most GnRH analogues lacked the carboxy-terminal Gly¹⁰-amide of GnRH and an ethylamide residue was added to Pro⁹, a modification common in many potent GnRH agonists, such as leuprolide ([D-Leu⁶, des-Gly¹⁰]-GnRH-NHEt. Results show differential impact of these modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation. ATPC in the N-terminus resulted in analogues with low binding affinity but high antiproliferative effect. Substitutions in position 6 always resulted in high binding affinities. In particular, [D-Lys⁶(Gly), desGly¹⁰]-GnRH-NHEt and [D-Lys⁶(Sar), desGly¹⁰]-GnRH-NHEt have higher pituitary binding affinity than leuprolide, but only the latter had significant antiproliferative effect on both MCF-7 and MDA-MB-231 cells. These results contribute to the on-going research for more potent GnRH analogues. Abbreviations of common amino acids are in accordance with the recommendations of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Arch. Biochem. Biophys. 206, pp.v-xxii (1988), J. Biol. Chem. 264, 668–673 (1989) or J. Peptide Sci. 9, 1–8 (2003).