Structural diversity in the type IV pili of multidrug-resistant Acinetobacter

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating thatA. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter. Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species

NMR Detection and Study of Hydrolysis of HNO-Derived Sulfinamides

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS­(O)­NH₂] is unique to HNO. We have applied ¹⁵N-edited ¹H NMR techniques to detect the HNO-induced thiol to sulfinamide modification in several small organic molecules, peptides, and the cysteine protease, papain. Relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS­(O)­OH]. We have investigated sulfinamide hydrolysis at physiological pH and temperature. Studies with papain and a related model peptide containing the active site thiol suggest that sulfinamide hydrolysis can be enhanced in a protein environment. These findings are also supported by modeling studies. In addition, analysis of peptide sulfinamides at various pH values suggests that hydrolysis becomes more facile under acidic conditions

Adding Diverse Noncanonical Backbones to Rosetta: Enabling Peptidomimetic Design

<div><p>Peptidomimetics are classes of molecules that mimic structural and functional attributes of polypeptides. Peptidomimetic oligomers can frequently be synthesized using efficient solid phase synthesis procedures similar to peptide synthesis. Conformationally ordered peptidomimetic oligomers are finding broad applications for molecular recognition and for inhibiting protein-protein interactions. One critical limitation is the limited set of design tools for identifying oligomer sequences that can adopt desired conformations. Here, we present expansions to the ROSETTA platform that enable structure prediction and design of five non-peptidic oligomer scaffolds (noncanonical backbones), oligooxopiperazines, oligo-peptoids, -peptides, hydrogen bond surrogate helices and oligosaccharides. This work is complementary to prior additions to model noncanonical protein side chains in ROSETTA. The main purpose of our manuscript is to give a detailed description to current and future developers of how each of these noncanonical backbones was implemented. Furthermore, we provide a general outline for implementation of new backbone types not discussed here. To illustrate the utility of this approach, we describe the first tests of the ROSETTA molecular mechanics energy function in the context of oligooxopiperazines, using quantum mechanical calculations as comparison points, scanning through backbone and side chain torsion angles for a model peptidomimetic. Finally, as an example of a novel design application, we describe the automated design of an oligooxopiperazine that inhibits the p53-MDM2 protein-protein interaction. For the general biological and bioengineering community, several noncanonical backbones have been incorporated into web applications that allow users to freely and rapidly test the presented protocols (<a href="http://rosie.rosettacommons.org" target="_blank">http://rosie.rosettacommons.org</a>). This work helps address the peptidomimetic community's need for an automated and expandable modeling tool for noncanonical backbones.</p></div

OOP ring conformations and validation of mm_std score function on side chain dihedrals in different ring conformations.

<p>A) Overlay of half-chair (blue) and boat (green) OOP ring conformations. B) Structure of half-chair OOP ring conformation. C) Structure of boat OOP ring conformation. D) QM energy calculations (Kcal/mol) of different side chain dihedral angles. Blue represents half-chair conformation. Green represents boat conformation. The half-chair conformation is lower in energy in two out of the three dihedral energy wells ( and ) but nearly isoenergetic with the boat conformation in the third (). E) QM energy (solid) and ROSETTA mm_std (dash, ROSETTA Energy Units) energy calculations for dihedral angles with OOP ring in half-chair conformation. Red X's show values of oxopiperazine side chains (labeled with Cambridge Structural Database code). F) QM energy (solid) and ROSETTA mm_std (dash, ROSETTA Energy Units) energy calculations for dihedral angles with OOP ring in boat conformation. For both (E) and (F), ROSETTA mm_std score function recapitulates the low energy minima.</p

OOP patch N-terminal section.

<p>Sample section of the N-terminal section of the OOP Pre patch file. Describes new atoms, bonds and internal coordinates as well as other patch specific parameter declarations. Full OOP Pre patch file can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067051#pone.0067051.s001" target="_blank">file S1</a>.</p

Demonstration of the NCBB Design Server Application.

<p>The OOP scaffold was designed to inhibit the p53-MDM2 protein interaction using the <b>NCBB Design</b> server. A) Crystal structure of p53 (pink sticks) - MDM2 (electrostatic surface) protein interaction with three hotspot residues highlighted (Phe19, Trp23, Leu26) which are responsible for majority of interaction's binding affinity (pdbid: 1YCR). B) Starting structure of alanine OOP scaffold (cyan) placed into the binding pocket of MDM2. C) Final designed structure reported by <b>NCBB Design</b> server. The final design shows a histidine designed in the first position recovering an aromatic residue similar to the hotspot Phe19. A tryptophan is designed in the second position recovering the hotspot residue of Trp23 and a Leu is designed in the fourth position recovering the hotspot residue Leu26.</p

Validation of mm_std score function on OOP dimer.

<p>Noncanonical backbones often require score functions that are based on molecular mechanics rather than the traditional ROSETTA knowledge based terms. A) Quantum mechanics (QM) and B) ROSETTA mm_std energy calculations of and torsion angle combinations for an OOP dimer (C). The and torsion angles are of the linker residue between two OOP rings and are labeled in C. Blue regions represent low energy (high probability) conformations, red regions represent high energy (low probability) conformations. The QM plot is measured in Kcals/mol while the mm_std plot is measured in ROSETTA Energy Units (REU). The ROSETTA mm_std calculations recover the main low energy wells as predicted by QM calculations with the lowest energy conformation estimated by QM marked by an ‘X’ on both plots. The structure in (C) is of the low energy conformation as predicted by QM.</p

Noncanonical backbone implementation flow chart.

<p>Outline of the five steps needed for implementation of a noncanonical backbone into ROSETTA.</p

Code snippet for correctly placing hydrogens on the OOP ring.

<p>After conformational changes of an OOP ring, hydrogens are often not in ideal positions. This code calculates a correction factor by determining the angle by which a virtual atom and a carbon atom across a cut point align. The torsion angle that defines the hydrogens movement is altered by this correction factor to properly align the hydrogens. A visual representation can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067051#pone.0067051.s003" target="_blank">movie S1</a>.</p