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Abstract Background Bacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing. Results Moraxella catarrhalis strain E22 containing plasmid pLQ510 was shown to inhibit the growth of M. catarrhalis strain O35E. Two genes (mcbA and mcbB) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (mcbC) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (mcbI) immediately downstream from mcbC encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the mcbI gene in M. catarrhalis O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the mcbA, mcbB, mcbC, and mcbI ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several M. catarrhalis strains including O12E. Inactivation of the mcbA, mcbB, or mcbC ORFs in M. catarrhalis O12E eliminated the ability of this strain to inhibit the growth of M. catarrhalis O35E. In co-culture experiments involving a M. catarrhalis strain containing the mcbABCI locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth. Conclusion This is the first description of a bacteriocin and its cognate immunity factor produced by M. catarrhalis. The killing activity of the McbC protein raises the possibility that it might serve to lyse other M. catarrhalis strains that lack the mcbABCI locus, thereby making their DNA available for lateral gene transfer.

The rational design of materials with cell-selective membrane activity is an effective strategy for the development of targeted molecular imaging and therapy. Here we report a new class of cationic multidomain peptides (MDPs) that can undergo enzyme-mediated molecular transformation followed by supramolecular assembly to form nanofibers in which cationic clusters are presented on a rigid β-sheet backbone. This structural transformation, which is induced by cells overexpressing the specific enzymes, led to a shift in the membrane perturbation potential of the MDPs, and consequently enhanced cell uptake and drug delivery efficacy. We envision the directed self-assembly based on modularly designed MDPs as a highly promising approach to generate dynamic supramolecular nanomaterials with emerging membrane activity for a range of disease targeted molecular imaging and therapy applications., (© 2021 Wiley-VCH GmbH.)

A small-scale ITC benchmarking study was performed involving 9 biophysics laboratories/facilities, to evaluate inter-laboratory and intra-laboratory basal levels of uncertainty. Our prime goal was to assess a number of important factors that can influence both the data gathered by this technique and the thermodynamic parameter values derived therefrom. In its first part, the study involved 5 laboratories and 13 different instruments, working with centrally prepared samples and the same experimental protocol. The second part involved 4 additional laboratories and 6 more instruments, where the users prepared their own samples according to provided instructions and did the experiments following the same protocol as in the first part. The study design comprised: (1) selecting a minimal set of laboratories; (2) providing very stable samples; (3) providing samples not requiring preparation or manipulation; and (4) providing a well-defined and detailed experimental protocol. Thus, we were able to assess: (i) the variability due to instrument and data analysis performed by each user on centrally prepared samples; (ii) the comparability of data retrieved when using 4 different software packages to analyze the same data, besides the data analysis carried out by the different users on their own experimental results; and (iii) the variability due to local sample preparation (second part of the study). Individual values, as well as averages and standard deviations for the binding parameters for EDTA-cation interaction, were used as metrics for comparing the equilibrium association constant (logK), enthalpy of interaction (ΔH), and the so-called "stoichiometry" (n), a concentration-correction factor.

Spectrins are tetrameric actin-cross-linking proteins that form an elastic network, termed the membrane skeleton, on the cytoplasmic surface of cellular membranes. At the plasma membrane, the membrane skeleton provides essential support, preventing loss of membrane material to environmental shear stresses. The skeleton also controls the location, abundance, and activity of membrane proteins that are critical to cell and tissue function. The ability of the skeleton to modulate membrane stability and function requires adaptor proteins that bind the skeleton to membranes. The principal adaptors are the ankyrin proteins, which bind to the beta-subunit of spectrin and to the cytoplasmic domains of numerous integral membrane proteins. Here, we present the crystal structure of the ankyrin-binding domain of human beta2-spectrin at 1.95 A resolution together with mutagenesis data identifying the binding surface for ankyrins on beta2-spectrin.

In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd approximately 350 microM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the Fe(III) form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.

Background: Biochemical and biophysical experiments have shown that two catalytically essential divalent metal ions (termed 'A' and 'B') bind to the 3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I. X-ray crystallographic studies have established the normal positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations and the single-stranded DNA substrate. Lanthanide (III) luminescence studies have demonstrated, however, that only a single europium (III) ion (Eu3+) binds to the KF exo active site. Furthermore, Eu3+ does not support catalysis by KF exo or several other two-metal-ion phosphoryl-transfer enzymes., Results: A crystal structure of KF complexed with both Eu3+ and substrate single-stranded oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A. Comparison of this structure to a relevant native structure reveals that the bound Eu3+ causes a number of changes to the KF exo active site. The scissile phosphate of the substrate is displaced from its normal position by about 1 A when Eu3+ is bound and the presence of Eu3+ in the active site precludes the binding of the essential metal ion B., Conclusions: The substantial, lanthanide-induced differences in metal-ion and substrate binding to KF exo account for the inhibition of this enzyme by Eu3+. These changes also explain the inability of KF exo to bind more than one cation in the presence of lanthanides. The mechanistic similarity between KF exo and other two-metal-ion phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion binding and inhibition ascertained from this study will probably apply to most members of this class of enzymes.

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.

A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments., (Copyright 1998 Academic Press Limited.off)

New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.