Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

Staphylococcus epidermidis and S. aureus are highly problematic bacteria in hospital settings. This stems, at least in part, from strong abilities to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized multicellular aggregates of bacteria, which, when formed on indwelling medical devices, lead to infections that are difficult to treat. Cell wall-anchored (CWA) proteins are known to be important players in biofilm formation and infection. Many of these proteins have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of the S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction or other significant conformational changes. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.

Impact of Heat on Coil Hydrodynamic Size Yields the Energetics of Denatured State Conformational Bias.

Conformational equilibria in the protein denatured state have key roles regulating folding, stability, and function. The extent of conformational bias in the protein denatured state under folding conditions, however, has thus far proven elusive to quantify, particularly with regard to its sequence dependence and energetic character. To better understand the structural preferences of the denatured state, we analyzed both the sequence dependence to the mean hydrodynamic size of disordered proteins in water and the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the α and polyproline II (PPII) backbone conformations can be obtained. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the protein reveal amino acid specific preferences for PPII that are in good quantitative agreement with calorimetry-measured values from unfolded peptides and those inferred by survey of the protein coil library. At temperatures above 25 °C, the denatured state follows the predictions of a PPII-dominant ensemble. Heat effects on coil hydrodynamic size indicate the α bias is comparable to the PPII bias at cold temperatures. Though historically thought to give poor resolution to structural details, the hydrodynamic size of the unfolded state is found to be an effective reporter on the extent of the biases for the α and PPII backbone conformations.

Sequence Reversal Prevents Chain Collapse and Yields Heat-Sensitive Intrinsic Disorder.

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of "Retro-nuclease" is highly expanded in solution with characteristics similar to biological intrinsically disordered proteins. In contradistinction to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hydrodynamic dimensions as temperature is increased, indicating a thermodynamically controlled compaction. Counterintuitively, the magnitude of these temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calorimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluorescence changes throughout the thermal transition indicate canonical hydrophobic collapse did not drive the Retro-nuclease structural transitions. Temperature-dependent circular dichroism spectroscopy performed on Retro-nuclease and computer simulations correlate to temperature sensitivity in the intrinsic sampling of backbone conformations for polyproline II and α-helix. The experimental results indicate a role for sequence direction in mediating the collapse of the polypeptide chain, whereas the simulation trends illustrate the generality of the observed heat effects on disordered protein structure.

Intrinsic α helix propensities compact hydrodynamic radii in intrinsically disordered proteins.

Proteins that lack tertiary stability under normal conditions, known as intrinsically disordered, exhibit a wide range of biological activities. Molecular descriptions for the biology of intrinsically disordered proteins (IDPs) consequently rely on disordered structural models, which in turn require experiments that assess the origins to structural features observed. For example, while hydrodynamic size is mostly insensitive to sequence composition in chemically denatured proteins, IDPs show strong sequence-specific effects in the hydrodynamic radius (Rh ) when measured under normal conditions. To investigate sequence-modulation of IDP Rh , disordered ensembles generated by a hard sphere collision model modified with a structure-based parameterization of the solution energetics were used to parse the contributions of net charge, main chain dihedral angle bias, and excluded volume on hydrodynamic size. Ensembles for polypeptides 10-35 residues in length were then used to establish power-law scaling relationships for comparison to experimental Rh from 26 IDPs. Results showed the expected outcomes of increased hydrodynamic size from increases in excluded volume and net charge, and compaction from chain-solvent interactions. Chain bias representing intrinsic preferences for α helix and polyproline II (PPII ), however, modulated Rh with intricate dependence on the simulated propensities. PPII propensities at levels expected in IDPs correlated with heightened Rh sensitivity to even weak α helix propensities, indicating bias for common (φ, ψ) are important determinants of hydrodynamic size. Moreover, data show that IDP Rh can be predicted from sequence with good accuracy from a small set of physicochemical properties, namely intrinsic conformational propensities and net charge. Proteins 2017; 85:296-311. © 2016 Wiley Periodicals, Inc.

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction.

Staphylococcus epidermidis is one of the primary bacterial species responsible for healthcare-associated infections. The most significant virulence factor for S. epidermidis is its ability to form a biofilm, which renders the bacteria highly resistant to host immune responses and antibiotic action. Intercellular adhesion within the biofilm is mediated by the accumulation-associated protein (Aap), a cell wall-anchored protein that self-assembles in a zinc-dependent manner. The C-terminal portion of Aap contains a 135-aa-long, proline/glycine-rich region (PGR) that has not yet been characterized. The region contains a set of 18 nearly identical AEPGKP repeats. Analysis of the PGR using biophysical techniques demonstrated the region is a highly extended, intrinsically disordered polypeptide with unusually high polyproline type II helix propensity. In contrast to many intrinsically disordered polypeptides, there was a minimal temperature dependence of the global conformational state of PGR in solution as measured by analytical ultracentrifugation and dynamic light scattering. Furthermore, PGR was resistant to conformational collapse or α-helix formation upon the addition of the osmolyte trimethylamine N-oxide or the cosolvent 2,2,2-trifluoroethanol. Collectively, these results suggest PGR functions as a resilient, extended stalk that projects the rest of Aap outward from the bacterial cell wall, promoting intercellular adhesion between cells in the biofilm. This work sheds light on regions of low complexity often found near the attachment point of bacterial cell wall-anchored proteins.

Fungal metabolite ophiobolin A as a promising anti-glioma agent: In vivo evaluation, structure-activity relationship and unique pyrrolylation of primary amines.

Glioblastoma, the most common form of malignant primary brain tumor, is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches based on apoptosis induction in cancer cells. Previously, a fungal secondary metabolite ophiobolin A was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, thus, offering an innovative strategy to combat this type of cancer. The current work describes the results of a preliminary evaluation of ophiobolin A in an in vivo glioblastoma model and its chemical derivatization to establish first synthetically generated structure-activity relationship. The synthetic work has also led to the discovery of a unique reaction of ophiobolin A with primary amines suggesting the possibility of pyrrolylation of lysine residues on its intracellular target protein(s).