Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

Hormonal regulation of sialic acid-binding (SAS) protein synthesis of rat uterus

Sialic acid binding proteins (SAS) of rat uteri have been found in all three stages of the estrous cycle. To study the control of synthesis of these proteins two different animal models were used I-immature female rats (25 d) where the hormones estradiol (E₂) and progesterone (P₄) were given separately and together, and II-adult female rats where hormone treatment commenced 14 days after ovariectomy. The data indicated that E₂ initiated the synthesis of SAS proteins in the immature animals, while P₄ could inhibit SAS synthesis, either given alone or together with E₂. However, prior priming of the rat with E₂ and subsequent administration of P₄ stimulated SAS protein synthesis.     

Sialic acid binding protein of human endometrium: Its regulation by steroids

In the present study, we have observed that sialic acid binding protein (SABP – a 54 kDa glycoprotein which was isolated from human endometrial scrapings taken at various stages of the menstrual cycle from normal cycling females and purified to apparent homogeneity and was earlier reported from this laboratory) was found in sufficiently detectable amount in the endometrium of normal cycling women whereas it was found in lesser amount in tissue from women who have recently entered the post-menopause stage. SABP was observed in both follicular and luteal phase of menstrual cycle which was found by western blot analysis. In the de-novo synthesis experiment, synthesis and secretion of SABP was found to be stimulated by estradiol (E₂) whereas progesterone (P₄) was found to have no significant stimulatory effect on it which was also confirmed by HEC cell culture. In the HEC cell culture, priming of cells with E₂ was found to influence the effect of P₄ on SABP when it was added 2 h after E₂ administration. This was observed by doing immunoprecipitation followed by SDS-PAGE and autoradiography. Hence this report clearly indicates that E₂ regulates the synthesis and secretion of 54 kDa SABP from human endometrium. How E₂ priming of endometrium influences the effect of P₄ on SABP has been discussed.     

Conserved charged amino acids are key determinants for fatty acid binding proteins (FABPs)-membrane interactions. A multi-methodological computational approach

Based on the analysis of the mechanism of ligand transfer to membranes employing in vitro methods, Fatty Acid Binding Protein (FABP) family has been divided in two subgroups: collisional and diffusional FABPs. Although the collisional mechanism has been well characterized employing in vitro methods, the structural features responsible for the difference between collisional and diffusional mechanisms remain uncertain. In this work, we have identified the amino acids putatively responsible for the interaction with membranes of both, collisional and diffusional, subgroups of FABPs. Moreover, we show how specific changes in FABPs’ structure could change the mechanism of interaction with membranes. We have computed protein–membrane interaction energies for members of each subgroup of the family, and performed Molecular Dynamics simulations that have shown different configurations for the initial interaction between FABPs and membranes. In order to generalize our hypothesis, we extended the electrostatic and bioinformatics analysis over FABPs of different mammalian genus. Also, our methodological approach could be used for other systems involving protein–membrane interactions.     

MD simulation of subtilisin BPN' in a crystal environment.

In this paper we present a molecular dynamics (MD) simulation of subtilisin BPN' in a crystalline environment containing four protein molecules and solvent. Conformational and dynamic properties of the molecules are compared with each other and with respect to the X-ray structure to test the validity of the force field. The agreement between simulated and experimental structure using the GROMOS force field is better than that obtained in the literature using other force fields for protein crystals. The overall shape of the molecule is well preserved, as is the conformation of alpha-helices and beta-strands. Structural differences are mainly found in loop regions. Solvent networks found in the X-ray structure were reproduced by the simulation, which was unbiased with respect to the crystalline hydration structure. These networks seem to play an important role in the stability of the protein; evidence of this is found in the structure of the active site. The weak ion binding site in the X-ray structure of subtilisin BPN' is occupied by a monovalent ion. When a calcium ion is placed in the initial structure, three peptide ligands are replaced by 5 water ligands, whereas a potassium ion retains (in part) its original ligands. Existing force fields yield a reliable method to probe local structure and short-time dynamics of proteins, providing an accuracy of about 0.1 nm.     

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer’s disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin – the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn²⁺-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.