PepComposer: computational design of peptides binding to a given protein surface

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver

The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein

Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years

Functional Analysis of MmeI from Methanol Utilizer Methylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes▿

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5′-TCCRAC-3′ (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N6-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D70-X9-EXK82, characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N6-adenine γ-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme

Co-translational binding of importins to nascent proteins

Abstract Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning

Passage of the HIV capsid cracks the nuclear pore

Upon infection, human immunodeficiency virus (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T-cells and macrophages. As its largest known cargo, the capsid enters the nuclear pore complex (NPC), driven by interactions with numerous FG-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryo electron tomography and molecular simulations to study nuclear entry of HIV-1 capsids in primary human macrophages. We found that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings