Biomedical subjects

Dimitrios Morikis

Publications and source records attributed to Dimitrios Morikis.

8 recordsLinked to original sources

PubMed2003

Conformational interconversion in compstatin probed with molecular dynamics simulations.

Compstatin is a 13-residue cyclic peptide that has the potential to become a therapeutic agent against unregulated complement activation. In our effort to understand the structural and dynamic characteristics of compstatin that form the basis for rational and combinatorial optimization of structure and activity, we performed 1-ns molecular dynamics (MD) simulations. We used as input in the MD simulations the ensemble of 21 lowest energy NMR structures, the average minimized structure, and a global optimization structure. At the end of the MD simulations we identified five conformations, with populations ranging between 9% and 44%. These conformations are as follows: 1) coil with alphaR-alphaR beta-turn, as was the conformation of the initial ensemble of NMR structures; 2) beta-hairpin with epsilon-alphaR beta-turn; 3) beta-hairpin with alphaR-alphaR beta-turn; 4) beta-hairpin with alphaR-beta beta-turn; and 5) alpha-helical. Conformational switch was possible with small amplitude backbone motions of the order of 0.1-0.4 A and free energy barrier crossing of 2-11 kcal/mol. All of the 21 MD structures corresponding to the NMR ensemble possessed a beta-turn, with 14 structures retaining the alphaR-alphaR beta-turn type, but the average minimized structure and the global optimization structures were converted to alpha-helical conformations. Overall, the MD simulations have aided to gain insight into the conformational space sampled by compstatin and have provided a measure of conformational interconversion. The calculated conformers will be useful as structural and possibly dynamic templates for optimization in the design of compstatin using structure-activity relations (SAR) or dynamics-activity relations (DAR).

Computer Simulation↗

PubMed2003

Studies of structure-activity relations of complement inhibitor compstatin.

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile(1)Leu/His(9)Trp/Thr(13)Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His(9)Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the beta-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His(9)Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.

Acetylation↗

PubMed2003

Related protein-protein interaction modules present drastically different surface topographies despite a conserved helical platform.

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.

Amino Acid Sequence↗

PubMed2003

Integrated computational and experimental approach for lead optimization and design of compstatin variants with improved activity.

A novel structure-activity-based combinatorial computational optimization methodology for the design of peptides that are candidates to become therapeutics is presented. This methodology has been successfully applied in the design of a 7-fold more active analogue, among other active analogues, in the case of the complement inhibitor compstatin. The main steps of the approach involve the availability of NMR-derived structural templates, combinatorial selection of sequences based on optimization of parametrized pairwise residue interaction potentials, prediction of fold stabilities using deterministic global optimization, and experimental validation with immunological activity measurements. This work is direct evidence that an integrated experimental and theoretical approach can make the engineering of compounds with enhanced immunological properties possible.

Amino Acid Sequence↗

PubMed2003

Compstatin, a peptide inhibitor of complement, exhibits species-specific binding to complement component C3.

Although activation of complement protein C3 is essential for the generation of normal inflammatory responses against pathogens, its unregulated activation during various pathological conditions leads to host cell damage. Previously we have identified a 13-residue cyclic peptide, Compstatin, that inhibits C3 activation. In this study, we have examined the species-specificity of Compstatin. Bimolecular interaction analysis using a real-time surface plasmon resonance-based assay showed that Compstatin exhibits exclusive specificity for primate C3s and does not bind either to C3s from lower mammalian species or to two structural homologs of C3, human C4 and C5. Furthermore, it showed that although the kinetics of binding of Compstatin to non-human primate C3s were distinctly different from those to human C3, like human C3 its mechanism of binding to non-human primate C3 was biphasic and did not follow a simple 1:1 interaction, suggesting that this binding mechanism could be important for its inhibitory activity. Analysis of Ala substitution analogs of Compstatin for their inhibitory activities against mouse and rat complement suggested that the lack of binding of Compstatin to mouse and rat C3s was not a result of sterically hindered access to the binding pocket due to individual bulky side chains or the presence of charge on the Compstatin molecule. These results suggest that Compstatin's exclusive specificity for primate C3s could be exploited for the development of species-specific complement inhibitors.

Amino Acid Substitution↗

PubMed2003

Complement: structure, functions, evolution, and viral molecular mimicry.

The complement (C') system has long been recognized as an important mediator of innate immune defense and inflammation. In recent years there is increasing evidence suggesting that complement components may also participate in non-inflammatory and developmental processes. Here we review our current work on the structural-functional aspects of C3-ligand interactions and the rational design of small-sized complement inhibitors. We present a novel, proteomics-based, approach to studying protein-protein interactions within the C' system and discuss our progress in the study of viral immune evasion strategies. Furthermore we discuss the involvement of complement proteins in organ regeneration and hematopoietic development.

Animals↗

PubMed2002

The structural basis of compstatin activity examined by structure-function-based design of peptide analogs and NMR.

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Amino Acid Sequence↗

PubMed2002

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.

Binding Sites↗