Sergey A. Samsonov

A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

We have identified a novel gene in a genome-wide, double-strand break DNA repair RNAi screen and show that is involved in the neurological disease hereditary spastic paraplegia.

Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.

Docking glycosaminoglycans to proteins: analysis of solvent inclusion

Glycosaminoglycans (GAGs) are anionic polysaccharides, which participate in key processes in the extracellular matrix by interactions with protein targets. Due to their charged nature, accurate consideration of electrostatic and water-mediated interactions is indispensable for understanding GAGs binding properties. However, solvent is often overlooked in molecular recognition studies. Here we analyze the abundance of solvent in GAG-protein interfaces and investigate the challenges of adding explicit solvent in GAG-protein docking experiments. We observe PDB GAG-protein interfaces being significantly more hydrated than protein–protein interfaces. Furthermore, by applying molecular dynamics approaches we estimate that about half of GAG-protein interactions are water-mediated. With a dataset of eleven GAG-protein complexes we analyze how solvent inclusion affects Autodock 3, eHiTs, MOE and FlexX docking. We develop an approach to de novo place explicit solvent into the binding site prior to docking, which uses the GRID program to predict positions of waters and to locate possible areas of solvent displacement upon ligand binding. To investigate how solvent placement affects docking performance, we compare these results with those obtained by taking into account information about the solvent position in the crystal structure. In general, we observe that inclusion of solvent improves the results obtained with these methods. Our data show that Autodock 3 performs best, though it experiences difficulties to quantitatively reproduce experimental data on specificity of heparin/heparan sulfate disaccharides binding to IL-8. Our work highlights the current challenges of introducing solvent in protein-GAGs recognition studies, which is crucial for exploiting the full potential of these molecules for rational engineering.

Position-dependent effects of fluorinated amino acids on the hydrophobic core formation of a heterodimeric coiled coil.

Systematic model investigations of the molecular interactions of fluorinated amino acids within native protein environments substantially improve our understanding of the unique properties of these building blocks. A rationally designed heterodimeric coiled coil peptide (VPE/VPK) and nine variants containing amino acids with variable fluorine content in either position a16 or d19 within the hydrophobic core were synthesized and used to evaluate the impact of fluorinated amino acid substitutions within different hydrophobic protein microenvironments. The structural and thermodynamic stability of the dimers were examined by applying both experimental (CD spectroscopy, FRET, and analytical ultracentrifugation) and theoretical (MD simulations and MM-PBSA free energy calculations) methods. The coiled coil environment imposes position-dependent conformations onto the fluorinated side chains and thus affects their packing and relative orientation towards their native interaction partners. We find evidence that such packing effects exert a significant influence on the contribution of fluorine-induced polarity to coiled coil folding.

A molecular dynamics approach to study the importance of solvent in protein interactions

Water constitutes the cellular environment for biomolecules to interact. Solvent is important for protein folding and stability, and it is also known to actively participate in many catalytic processes in the cell. However, solvent is often ignored in molecular recognition and not taken into account in protein–protein interaction studies and rational design. Previously we developed SCOWLP, a database and its web application (http://www.scowlp.org), to perform studies on the contribution of solvent to protein interface definition in all protein complexes of the PDB. We introduced the concept of wet spots, interfacial residues interacting only through one water molecule, which were shown to considerably enrich protein interface descriptions. Analysis of interfacial solvent in a nonredundant dataset of protein complexes suggested the importance of including interfacial water molecules in protein interaction studies. In this work we use a molecular dynamics approach to gain deeper insights into solvent contribution to protein interfaces. We characterize the dynamic and energetic properties of water‐mediated protein interactions by comparing different interfacial interaction types (direct, dual and wet spot) at residue and solvent level. For this purpose, we perform an analysis of 17 representative complexes from two protein families of different interface nature. Energetically wet spots are quantitatively comparable to other residues in interfaces, and their mobility is shown to be lower than protein surface residues. The residence time of water molecules in wet spots sites is higher than of those on the surface of the protein. In terms of free energy, though wet‐spots‐forming water molecules are very heterogeneous, their contribution to the free energy of complex formation is considerable. We find that water molecules can play an important role in interaction conservation in protein interfaces by allowing sequence variability in the corresponding binding partner, and we discuss the important implications of our observations related to the use of the correlated mutations concept in protein interactions studies. The results obtained in this work help to deepen our understanding of the physico–chemical nature underlying protein–protein interactions and strengthen the idea of using the wet spots concept to qualitatively improve the accuracy of folding, docking and rational design algorithms. Proteins 2008.

Sulfated Glycosaminoglycans Exploit the Conformational Plasticity of Bone Morphogenetic Protein-2 (BMP-2) and Alter the Interaction Profile with Its Receptor

Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.

Binding of chondroitin 4-sulfate to cathepsin S regulates its enzymatic activity.

Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 μM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.

Fluorine in Protein Environments: A QM and MD Study

Noncanonical amino acids with newly designed side-chain functionalities represent powerful tools to improve structural, biological, and pharmacological properties of peptides and proteins. In this context, fluorinated amino acids have increasingly gained importance. Despite the current wide use of fluorination in protein engineering, the basic properties of fluorine in protein environments are still not completely understood. Our aim has been to characterize the physicochemical properties of fluorinated amino acids by using quantum mechanics (QM) and molecular dynamics (MD) approaches. We have analyzed geometry, charges, and hydrogen bonding abilities of several ethane fluorinated derivatives at different QM theory levels and have used them as simplified models for fluorinated amino acid side chains. We have parametrized four fluorinated L-amino acids for the AMBER ff94/99 force field: 4-monofluoroethylglycine (MfeGly), 4,4-difluoroethylglycine (DfeGly), 4,4,4-trifluoroethylglycine (TfeGly), and 4,4-difluoropropylglycine (DfpGly). We have characterized them in terms of molecular volumes, conformational preferences, and hydration properties. The obtained results illustrate that fluorine and hydrogen atoms of fluoromethyl groups could be potential acceptors or donors of weak hydrogen bonds in protein environments. Hydration of the studied fluorinated amino acids was found to be more favorable than for their nonfluorinated analogues, and hydrophobicity was observed to increase with the number of fluorine atoms, which is in accordance with the experimental retention times we obtain for these amino acids. This study broadens our understanding of the properties of fluorine within protein environments, which is important to exploit the full potential of fluorines unique properties for applications in the field of protein engineering.

Artificial extracellular matrix composed of collagen I and highly sulfated hyaluronan interferes with TGFβ1 signaling and prevents TGFβ1-induced myofibroblast differentiation

Sulfated glycosaminoglycans are promising components for functional biomaterials since sulfate groups modulate the binding of growth factors and thereby influence wound healing. Here, we have investigated the influence of an artificial extracellular matrix (aECM) consisting of collagen I (coll) and hyaluronan (HA) or highly sulfated HA (hsHA) on dermal fibroblasts (dFb) with respect to their differentiation into myofibroblasts (MFb). Fibroblasts were cultured on aECM in the presence of aECM-adsorbed or soluble transforming growth factor β1 (TGFβ1). The synthesis of α-smooth muscle actin (αSMA), collagen and the ED-A splice variant of fibronectin (ED-A FN) were analyzed at the mRNA and protein levels. Furthermore, we investigated the bioactivity and signal transduction of TGFβ1 in the presence of aECM and finally made interaction studies of soluble HA or hsHA with TGFβ1. Artificial ECM composed of coll and hsHA prevents TGFβ1-stimulated αSMA, collagen and ED-A FN expression. Our data suggest an impaired TGFβ1 bioactivity and downstream signaling in the presence of aECM containing hsHA, shown by massively reduced Smad2/3 translocation to the nucleus. These data are explained by in silico docking experiments demonstrating the occupation of the TGFβ-receptor I binding site by hsHA. Possibly, HA sulfation has a strong impact on TGFβ1-driven differentiation of dFb and thus could be used to modulate the properties of biomaterials.

Flexibility and explicit solvent in molecular-dynamics-based docking of protein-glycosaminoglycan systems.

We present Dynamic Molecular Docking (DMD), a novel targeted molecular dynamics-based protocol developed to address ligand and receptor flexibility as well as the inclusion of explicit solvent in local molecular docking. A class of ligands for which docking performance especially benefits from overcoming these challenges is the glycosaminoglycans (GAGs). GAGs are periodic, highly flexible, and negatively charged polysaccharides playing an important role in the extracellular matrix via interaction with proteins such as growth factors and chemokines. The goal of our work has been to develop a proof of concept for an MD-based docking approach and to analyze its applicability for protein-GAG systems. DMD exploits the electrostatics-driven attraction of a ligand to its receptor, treats both as entirely flexible, and considers solvent explicitly. We show that DMD has high predictive significance for systems dominated by electrostatic attraction and demonstrate its capability to reliably identify the receptor residues contributing most to binding.