Biology subjects

Loll, B.

Publications and source records attributed to Loll, B..

6 recordsLinked to original sources

bioRxiv2022

Gene amplifications cause high-level resistance against albicidin in Gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public health care. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against the antibiotic albicidin. We show that this mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of the GyrI-like transcription regulator STM3175 (YgiV) which binds albicidin. X-ray crystallography and molecular docking studies reveal a conserved binding motif that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in Gram-negative bacteria and our experiments confirm that STM3175 homologs can convey resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

microbiology↗

bioRxiv2022

Water network in the binding pocket of fluorinated BPTI-Trypsin complexes - insights from simulation and experiment

Structural waters in the S1 binding pocket of {beta}-trypsin are critical for the stabilization of the complex of {beta}-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by -aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly) and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific non-covalent interactions to the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly-BPTI-{beta}-trypsin complex (pdb: 7PH1). Both, experimental and computational data, show a step-wise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regards to energetic properties that could explain the increase in inhibitor strength. The analysis of the enthalpy and the entropy of the water molecules in the S1 binding pocket using Grid Inhomogeneous Solvation Theory confirms this result. Overall, fluorination systematically changes the binding affinity but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly. TOC GRAPHIC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/496563v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1177443org.highwire.dtl.DTLVardef@d20760org.highwire.dtl.DTLVardef@e252ecorg.highwire.dtl.DTLVardef@b22121_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development. Author summaryEquine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) are endemic in horses and cause great suffering as well as substantial economic losses to the equine industry. Current vaccines do not prevent infections and treatment is difficult. A prerequisite for vaccine and drug development is an in-depth understanding of the virus replication cycle, especially the virus entry process in order to block the infection at early stages. Entry of alphaherpesviruses into the host cell is mediated by a set of virus envelope glycoproteins including glycoprotein D (gD) that triggers the internalization of the virus particle. The structure of gD and the interaction with the entry receptor equine major histocompatibility complex class I (MHC-I) remains elusive. Here, we solved the crystal structures of gD1 and gD4 that allowed us to model virus-receptor interaction and to determine the key residues for virus entry. Alterations of these key residues impaired virus growth in cell culture. The overall structure of gD1 and gD4 shows classical features of other alphaherpesvirus gDs making it possible to gain further insights into human pathogens as well.

molecular biology↗

bioRxiv2021

Molecular Basis of Antibiotic Self-Resistance in a Bee Larvae Pathogen

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays high antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in P. larvae by deactivation of paenilamicin. Using tandem MS, NMR spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a new ribosome inhibitor. Here, we further elucidated the crystal structure of PamZ:acetyl-CoA complex at 1.34 [A] resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of P. larvae to fight American Foulbrood.

biochemistry↗

bioRxiv2021

The superior salinity tolerance of wheat cultivar Shanrong No. 3 cannot be attributed to elevated Ta-sro1 poly(ADP-ribose) polymerase activity

Saline soils limit the production of important staple crops such as wheat, particularly in arid and semiarid regions. Salt tolerance is a multi-gene trait and this complicates breeding of wheat varieties that deliver high yields under saline soil conditions. Notably, the elevated salinity tolerance of wheat cultivar Shanrong No. 3 (SR3) has been linked to a specific proteoform of the wheat SIMILAR TO RCD1 ONE (SRO1) protein that was created in an asymmetric genome hybridization with tall wheat grass. The two amino acid polymorphisms of the Ta-sro1 proteoform enhance the poly(ADP-ribose) polymerase (PARP) activity of the protein suggesting that altered poly-ADP-ribosylation of unknown substrate proteins or nucleic acids underlie the elevated salinity tolerance of cultivar SR3. To elucidate the molecular basis for the elevated PARP activity of the Ta-sro1 proteoform we solved a crystal structure of the catalytic PARP domain. Surprisingly, the structure revealed that the postulated binding site for the co-substrate NAD+ substantially differs from the structurally conserved NAD+ binding sites of canonical PARP enzymes. Consistently, we find that Ta-sro1 does not bind NAD+ and lacks ADP-ribosyltransferase activity. Therefore, although the structure revealed that one of the polymorphic amino acids is located close to the proposed active site, the elevated salinity tolerance of cultivar SR3 cannot be explained by altered ADP-ribosyltransferase activity of Ta-sro1.

plant biology↗

bioRxiv2021

Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 [A] resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

biophysics↗