Solution structure of regioselectively addressable functionalized templates: An NMR and restrained molecular dynamics investigation

Three cyclic peptides that are Regioselectively Addressable Functionalized Templates (RAFT) for use in protein de novo design have been investigated using a combination of nmr, restrained molecular dynamics, and CD spectroscopy. These peptides contain up to four selectively addressable sites (orthogonally protected lysine side chains) or have selectively addressable faces. The results show a common stable conformation for templates of this kind based on two type II β-turns and an associated β-sheet structure. A preferential orientation for the side chains is also demonstrated. The significance of these findings is discussed in the context of applications of RAFT that rely on their conformational rigidity and ability to present functionalities in a defined spatial arrangement. © 1996 John Wiley & Sons, Inc.     

Molecular modeling studies of substrate binding by penicillin acylase

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.     

Application of biasing‐potential replica‐exchange simulations for loop modeling and refinement of proteins in explicit solvent

Comparative or homology modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. Depending on the target‐template similarity these model structures may contain regions of limited structural accuracy. In principle, molecular dynamics (MD) simulations can be used to refine protein model structures and also to model loop regions that connect structurally conserved regions but it is limited by the currently accessible simulation time scales. A recently developed biasing potential replica exchange (BP‐REMD) method was used to refine loops and complete decoy protein structures at atomic resolution including explicit solvent. In standard REMD simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. In a BP‐REMD simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. The method requires much fewer replicas for efficient sampling compared with T‐REMD. Application of the approach to several protein loops indicated improved conformational sampling of backbone dihedral angle of loop residues compared to conventional MD simulations. BP‐REMD refinement simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional MD simulations. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

On the use of structural templates for high‐resolution docking

Reliable high‐resolution prediction of protein complex structures starting from the free monomers is a considerable challenge toward large‐scale mapping of the structural details of protein‐protein interactions. The current major bottleneck is to model the conformational changes of the monomer backbone upon binding. We evaluate the use of homolog structures as source for conformational diversity, within the framework of RosettaDock—a leading high‐resolution docking protocol. We find that the use of homolog templates can improve significantly the modeling of a complex structure, including known difficult cases. Several conformational changes however are not sampled by any of the templates, indicating the need for additional sources of conformational variability. Interestingly, the successful homolog templates are not restricted to a confined range of sequence identity, highlighting the importance of the backbone conformation rather than the sequence. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Molecular modeling and statistical analysis in the design of derivatives of human dipeptidyl peptidase IV

Human dipeptidyl peptidase IV (hDDP-IV) has a considerable importance in inactivation of glucagon-like peptide-1, which is related to type 2 diabetes. One approach for the treatment is the development of small hDDP-IV inhibitors. In order to design better inhibitors, we analyzed 5-(aminomethyl)-6-(2,4-dichlrophenyl)-2-(3,5-dimethoxyphenyl)pyrimidin-4-amine and a set of 24 molecules found in the BindingDB web database for model designing. The analysis of their molecular properties allowed the design of a multiple linear regression model for activity prediction. Their docking analysis allowed visualization of the interactions between the pharmacophore regions and hDDP-IV. After both analyses were performed, we proposed a set of nine molecules in order to predict their activity. Four of them displayed promising activity, and thus, had their docking performed, as well as, the pharmacokinetic and toxicological study. Two compounds from the proposed set showed suitable pharmacokinetic and toxicological characteristics, and therefore, they were considered promising for future synthesis and in vitro studies.     

Molecular modeling of the structures of human and rat pancreatic cholesterol esterases.

Structural models have been generated for rat and human cholesterol esterases by molecular modeling. For rat cholesterol esterase, three separate models were generated according to the following procedure: (1) the cholesterol esterase sequence was aligned with those of three template enzymes: Torpedo californica acetylcholinesterase, Geotrichum candidum lipase and Candida rugosa lipase; (2) the X-ray structure coordinates of the three template enzymes were used to construct cholesterol esterase models by amino acid replacements of matched sequence positions and by making sequence insertions and deletions as required; (3) bad contracts in each of the cholesterol esterase models were relaxed by molecular dynamics and mechanics; (4) the three cholesterol esterase models were merged into one by arithmetic averaging of atomic coordinates; (5) Ramachandran analysis indicated that the model generated from the AChE template possessed the best set of phi/psi angles. Therefore, this model was subjected to molecular dynamics, with harmonic constraints imposed on the C(alpha) coordinates to drive them toward the coordinates of the averaged model. (6) Subsequent relaxation by molecular mechanics produced the final rat cholesterol esterase model. A model for human cholesterol esterase was produced by repeating steps 1-3 above, albeit with the rat cholesterol esterase model as the template. Hydrophobic and electrostatic analyses of the rat and human cholesterol esterase models suggest the structural origins of molecular recognition of hydrophobic substrates and interfaces, of charged interfaces, and of bile salt activators.     

The combination of two‐dimensional and three‐dimensional analysis methods contributes to the understanding of glioblastoma spatial heterogeneity

Heterogeneity is regarded as the major factor leading to the poor outcomes of glioblastoma (GBM) patients. However, conventional two‐dimensional (2D) analysis methods, such as immunohistochemistry and immunofluorescence, have limited capacity to reveal GBM spatial heterogeneity. Thus, we sought to develop an effective analysis strategy to increase the understanding of GBM spatial heterogeneity. Here, 2D and three‐dimensional (3D) analysis methods were compared for the examination of cell morphology, cell distribution and large intact structures, and both types of methods were employed to dissect GBM spatial heterogeneity. The results showed that 2D assays showed only cross‐sections of specimens but provided a full view. To visualize intact GBM specimens in 3D without sectioning, the optical tissue clearing methods CUBIC and iDISCO+ were used to clear opaque specimens so that they would become more transparent, after which the specimens were imaged with a two‐photon microscope. The 3D analysis methods showed specimens at a large spatial scale at cell‐level resolution and had overwhelming advantages in comparison to the 2D methods. Furthermore, in 3D, heterogeneity in terms of cell stemness, the microvasculature, and immune cell infiltration within GBM was comprehensively observed and analysed. Overall, we propose that 2D and 3D analysis methods should be combined to provide much greater detail to increase the understanding of GBM spatial heterogeneity.     

Molecular modeling of flexible arm‐mediated interactions between bacterial chemoreceptors and their modification enzyme

Sensory adaptation in bacterial chemotaxis is mediated by methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of chemoreceptors. Methylation is catalyzed by methyltransferase CheR. In E. coli and related organisms, methylation sufficiently rapid to be physiologically effective requires a carboxyl terminal pentapeptide sequence on the receptor being modified or, via adaptational assistance, on a neighboring homodimer in a receptor cluster. Pentapeptide‐enhanced methylation is thought to be mediated by a ～30 residue, potentially disordered sequence that serves as a flexible arm connecting the receptor body and pentapeptide‐bound methyltransferase, thus allowing diffusionally restricted enzyme to reach methyl‐accepting sites. However, it was not known how many or which sites on the same or neighboring receptors were accessible to the tethered enzyme. We investigated using molecular modeling and found that, in a hexagonal array of trimers of receptor dimers, CheR tethered to a dimer of chemoreceptor Tar by its native 30‐residue flexible‐arm sequence could reach all methyl‐accepting sites on the dimer to which it was tethered plus 48 methyl‐accepting sites distributed among nine neighboring dimers, equivalent to the total sites carried by six receptors. This modeling‐determined methylation neighborhood of one enzyme‐binding dimer and six neighbors corresponds precisely with the experimentally identified neighborhood of seven. Thus, the experimentally observed adaptational assistance can occur by docking of pentapeptide‐bound, diffusionally restricted enzyme to methyl‐accepting sites on neighboring receptors. Our analysis introduces the notion that physiologically relevant adaptational assistance could occur even if only a subset of sites on a particular receptor are within reach.     

Two‐dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three‐dimensional crystals

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two‐dimensional (2D) layers within the context of three‐dimensional (3D) crystals. Here, we show that well‐ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His‐tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.     

Molecular modeling and multi‐spectroscopic approaches to study the interaction between antibacterial drug and human immunoglobulin G

Mechanistic and conformational studies on the interaction of sulfamethoxazole (SMX) with human immunoglobulin G (HIgG) were performed by molecular modeling and multi‐spectroscopic methods. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HIgG. The binding parameters and thermodynamic parameters at different temperatures had been calculated according to the Stern?Volmer, Scatchard, Sips and Van ’t Hoff equations, respectively. Experimental results showed that the fluorescence intensity of HIgG was quenched by the gradual addition of SMX. The binding constants of SMX with HIgG decreased with the increase of temperature, which meant that the quenching mechanism was a static quenching. Meanwhile, the results also confirmed that there was one independent class of binding site on HIgG for SMX during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?14.69 kJ·mol^?1 and 22.99 J·mol^?1·K^?1, respectively, which suggested that the electrostatic and hydrophobic interactions were the predominant intermolecular forces in stabilizing the SMX?HIgG complex. Furthermore, experimental results obtained from three‐dimensional fluorescence spectroscopy, UV?vis absorption spectroscopy and circular dichroism (CD) spectroscopy confirmed that the conformational structure of HIgG was altered in the presence of SMX. Copyright © 2015 John Wiley & Sons, Ltd.     

Molecular modeling reveals binding interface of γ‐tubulin with GCP4 and interactions with noscapinoids

The initiation of microtubule assembly within cells is guided by a cone shaped multi‐protein complex, γ‐tubulin ring complex (γTuRC) containing γ‐tubulin and atleast five other γ‐tubulin‐complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ‐tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/β?tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ‐tubulin and GCP4 through computational techniques. We simulated two complexes of γ‐tubulin‐GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of ?158.82 and ?170.19 kcal/mol for dimer1 and ?79.53 and ?101.50 kcal/mol for dimer2 using MM‐PBSA and MM‐GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ‐tubulin. From the results of the free‐energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ‐tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ‐tubulin‐GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ‐tubulin‐GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo‐noscapine followed by noscapine and amino‐noscapine. This offers a novel chemical scaffold for γ‐tubulin binding drugs near γ‐tubulin‐GCP4 interface. Proteins 2015; 83:827–843. © 2015 Wiley Periodicals, Inc.     

Beyond carbon and nitrogen: guidelines for estimating three‐dimensional isotopic niche space

Isotopic niche has typically been characterized through carbon and nitrogen ratios and most modeling approaches are limited to two dimensions. Yet, other stable isotopes can provide additional power to resolve questions associated with foraging, migration, dispersal and variations in resource use. The ellipse niche model was recently generalized to n‐dimensions. We present an analogous methodology which incorporates variation across three stable dimensions to estimate the significant features of a population's isotopic niche space including: 1) niche volume (referred to as standard ellipsoid volume, SEV), 2) relative centroid location (CL), 3) shape and 4) area of overlap between multiple ellipsoids and 5) distance between two CLs. We conducted a simulation study showing the accuracy and precision of three dimensional niche models across a range of values. Importantly, the model correctly identifies differences in SEV and CL among populations, even with small sample sizes and in cases where the absolute values cannot precisely be recovered. We use these results to provide guidelines for sample size in conducting multivariate isotopic niche modeling. We demonstrate the utility of our approach with a case study of three bottlenose dolphin populations which appear to possess largely overlapping niches when analyzed with only carbon and nitrogen isotopes. Upon inclusion of sulfur, we see that the three dolphin ecotypes are in fact segregated on the basis of salinity and find the stable isotope niche of inshore bottlenose dolphins significantly larger than coastal and offshore populations.     

X‐ray vs. NMR structures as templates for computational protein design

Certain protein‐design calculations involve using an experimentally determined high‐resolution structure as a template to identify new sequences that can adopt the same fold. This approach has led to the successful design of many novel, well‐folded, native‐like proteins. Although any atomic‐resolution structure can serve as a template in such calculations, most successful designs have used high‐resolution crystal structures. Because there are many proteins for which crystal structures are not available, it is of interest whether nuclear magnetic resonance (NMR) templates are also appropriate. We have analyzed differences between using X‐ray and NMR templates in side‐chain repacking and design calculations. We assembled a database of 29 proteins for which both a high‐resolution X‐ray structure and an ensemble of NMR structures are available. Using these pairs, we compared the rotamericity, χ₁‐angle recovery, and native‐sequence recovery of X‐ray and NMR templates. We carried out design using RosettaDesign on both types of templates, and compared the energies and packing qualities of the resulting structures. Overall, the X‐ray structures were better templates for use with Rosetta. However, for ～20% of proteins, a member of the reported NMR ensemble gave rise to designs with similar properties. Re‐evaluating RosettaDesign structures with other energy functions indicated much smaller differences between the two types of templates. Ultimately, experiments are required to confirm the utility of particular X‐ray and NMR templates. But our data suggest that the lack of a high‐resolution X‐ray structure should not preclude attempts at computational design if an NMR ensemble is available. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

Polarization‐dependent second‐harmonic generation (P‐SHG) microscopy is used to characterize molecular nonlinear optical properties of collagen and determine a three‐dimensional (3D) orientation map of collagen fibers within a pig tendon. C₆ symmetry is used to determine the nonlinear susceptibility tensor components ratios in the molecular frame of reference and , where the latter is a newly extracted parameter from the P‐SHG images and is related to the chiral structure of collagen. The is observed for collagen fibers tilted out of the image plane, and can have positive or negative values, revealing the relative polarity of collagen fibers within the tissue. The P‐SHG imaging was performed using a linear polarization‐in polarization‐out (PIPO) method on thin sections of pig tendon cut at different angles. The nonlinear chiral properties of collagen can be used to construct the 3D organization of collagen in the tissue and determine the orientation‐independent molecular susceptibility ratios of collagen fibers in the molecular frame of reference.      

Development by three‐dimensional approaches and four‐dimensional imaging: To the knowledge frontier and beyond

Many advances have been taken on elucidating embryonic development and tissue homeostasis and repair by the use of experimental strategies that preserve the three‐dimensional (3D) organization and allow quantitative analysis of images over time (four‐dimensional). Ranging from the understanding about the relationship between blastomeres and the events that take place during gastrulation by the use of time‐lapse imaging through 3D cultures that mimic organogenesis, the advances in this area are of critical value. The studies on embryonic development without disrupting the original architecture and the development of 3D organoid cultures pave a new avenue for unprecedented experimental advances that will positively impact the emergence of new treatments applying regenerative principles for both tissue repair and organ transplant. Birth Defects Research (Part C) 105:1–8, 2015. © 2015 Wiley Periodicals, Inc.     

Scaffold‐free inkjet printing of three‐dimensional zigzag cellular tubes

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160. © 2012 Wiley Periodicals, Inc.     

Accurate prediction of interfacial residues in two‐domain proteins using evolutionary information: Implications for three‐dimensional modeling

With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure–function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two‐domain proteins. We also use information from the three‐dimensional structures of individual domains of two‐domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (～85%) and specific (～95%) to the domain–domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain–domain interaction, rigid‐body docking was able to provide us with accurate full‐length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions. Proteins 2014; 82:1219–1234. © 2013 Wiley Periodicals, Inc.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.