Human Epidermal Growth Factor Receptor 2 (HER2) is, among EGFR family, one of the most relevant members as it remains overexpressed on tumor cells and provides resistance to well-studied anti-HER2 monoclonal antibody, Trastuzumab (Herceptin®), or tyrosine kinase inhibitor. Furthermore, HER2 plays a key role in the HER family due the interaction with other HER receptors via a complex signaling network to regulate cell growth, differentiation and survival. In this work, we have employed computational modelling and Molecular Dynamic (MD) simulations to attain a deeper understanding of the interaction of specific anti-HER2 antibodies and HER2. The dynamic behavior of HER2 receptor in complex with F0178 and scFv from Trastuzumab was investigated by two replicas of 0.5 µs MD simulations for each system as well as for the individual ones. A variety of structural, energetic and dynamic characteristics ranging from pairwise interactions formation to covariance analyses were performed to the 2 bundle complexes. Our aim was to understand the all-atom details of these intermolecular couplings, fundamental for the development of new therapies.

pH is one of the most important solution parameters. It plays a major role in most biochemical processes by, among other, inducing protein conformational changes and influencing protein-lipid interactions. These systems have been modeled using constant-pH molecular dynamics (CpHMD) methods since they are able to correctly capture the conformational/protonation coupling. In a previous CpHMD study, we have shown that, upon membrane insertion, the titrable amino acids are prone to adopt a neutral state. In that work, CpHMD experienced difficulties sampling ionized conformations in inserted regions, since in the time scale of our simulations most residues retained their neutral state upon insertion/desolvation.

Enhanced sampling techniques are a widely used solution to deal with kinetic traps in molecular dynamics simulations. Since our sampling problems are related with protonation, we have implemented a pH-based replica exchange (pHRE). In this method, each simulation replica is assigned a unique pH value and attempts to exchange the simulated pH value of simulations pairs are periodically performed. The acceptance criterion is influenced by the exchanging simulations pH values and protonation states of the titrable sites.

In this work, a more accurate description of the membrane influence on the pKa profiles of titrable amino acids is provided by using the pHRE methodology, a newly developed method to calculate insertion, and more rigorous criteria to define the acceptable protonation sampling. Since in pHRE, due to replica mixing, all pH values sample similar insertion regions, a larger amount of inserted conformations in the ionized state are obtained. Our efficient pHRE results outperformed previous CpHMD ones, granting more sampling in less simulation time. In the future, pHRE will replace CpHMD as our go-to method to study pH-dependent phenomena.

Hoechst 33342 (H33342) is a fluorescent probe that stains the DNA of living cells, permeating through cell membranes.[1] However, the influence of the probe ionization state [2] in this process is poorly characterized. The knowledge of H33342 ionization state in lipid bilayers will help to predict and interpret its passive permeation through cell membranes. In this work we characterized the acid/base properties of the interaction of H33342 with POPC bilayers using an experimental and computational combined approach.
H33342 pKa values in aqueous solution of 6.4 and 11.1 were measured by its UV/Visible spectra at different pH. H33342 partition coefficient (Kp) to POPC bilayers at different pH was measured by isothermal titration calorimetry (ITC). An increase of Kp for higher pH values was obtained, indicating stronger interaction with membranes for the less charged or neutral forms of the probe. The enthalpy variation (ΔH⁰) for the partition to the bilayer was negative at all pH values, with higher absolute values at low pH. This may indicate that when H33342 is more protonated, it adopts a more external position in the bilayer, being able to make favorable interactions in this membrane region. Detailed characterization of H33342-membrane interactions was also obtained through Molecular Dynamics (MD) simulations. This allowed to support experimental results by the calculation of membrane transverse location and preferential orientation of the H33342 in different protonation states, as well as possibility of hydrogen bonding between the probe and the membrane.
We conclude that at physiological pH H33342 presents a high fraction of the neutral form while associated with POPC bilayers, justifying the fast permeation observed through cell membranes.
Acknowledgements:
This work was partially supported by the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT) through projects 007630 UID/QUI/00313/2013 and PT2020_PTDC_DTP-FTO_2784_2014, cofunded by COMPETE2020-UE.

Protein-protein interactions (PPIs) are the foundation of basic organism functions and understanding them is key in determining the importance of different proteins in a wide array of complex networks and processes [1]. The variety underlying PPIs is immense and some residues are more essential in interface stabilization than others [2]. Such is the case of hot-spots (HS), residues whose mutation to alanine is detrimental for the stability of the PPI, as opposed to null-spots (NS), which constitute the remaining interfacial residues [3]. Considering the complex landscape in protein interfaces, some patterns and characteristics arise when a high amount of data is considered, by minimizing the effect of less prevalent interactions and characteristics. In this work, the SpotOn pipeline [4] - developed by our group - custom scripts and conservation servers were used to determine structural features of interfacial residues and to classify them as HS and NS in the PPI4DOCK database [5], comprising over 1400 non-redundant complexes. This study allowed us to further understand the structural differences between HS and NS and will be available in a web-server in the near future.

References

1. Jones, S. and J.M. Thornton, Principles of protein-protein interactions. Proc Natl Acad Sci USA, 1996. 93.
2. Stites, W.E., Protein-Protein Interactions: Interface Structure, Binding Thermodynamics, and Mutational Analysis. Chem Rev, 1997. 97(5): p. 1233-1250.
3. Bogan, A.A. and K.S. Thorn, Anatomy of hot spots in protein interfaces. J Mol Biol, 1998. 280(1): p. 1-9.
4. Moreira, I.S., et al., SpotOn: High Accuracy Identification of Protein-Protein Interface Hot-Spots. Scientific Reports, 2017. 7(1): p. 8007.
5. Yu, J. and R. Guerois, PPI4DOCK: large scale assessment of the use of homology models in free docking over more than 1000 realistic targets. Bioinformatics, 2016. 32(24): p. 3760-3767.

Due to the clinical importance of the Dopamine D2-receptor (D2R) in several brain dysfunctions, the utilization of in silico models for drug development is a growing field of investigation. We provided a transparent and reproducible pipeline for creating a valid D2R model for small molecular docking studies. Furthermore, we suggested a binding pocket for the endogenous ligand of D2R, which was attained upon careful consideration of the available experimental data. Molecular docking studies with Dopamine, Quinpirole and Raclopride allowed also a better understanding of the binding pocket characteristics.

Ghrelin is a peptide secreted in the gastric fundus and acts in hypophysis and hippocampus through a Class A G-protein Coupled Receptor (GPCR), the GHS-R1a. GPCRs are characterized by a seven transmembrane (TM) spanning α-helices, connected to three extracellular (ECL) and three intracellular loops (ICL). (1) Activation of this receptor is involved in a variety of functions from feeding and growth hormone release to promotion of learning and memory. Among GPCRs, GHS-R1a is characterized by an unusual high constitutive activity, in the absence of specific ligands. (2) Regarding this distinctive characteristic, a mutation, A204E, of this receptor leads to a reduction of its constitutive activity. (3)

To reveal more information about this distinctive receptor, 3D structure models of different stages of activation and of a particular mutation (A204E) of the GHS-R1a were created using homology modelling molecular docking  and their differences analysed. Fully-atomistic molecular dynamics simulations are being performed initially with the pre-active model and the mutant, and will be followed by the remaining activation states. These simulations will give atomic detail to the necessary conformation rearrangements responsible for receptor activity. The structural and functional characterization of GHSR1a is an important step towards the design of specific drugs.

1,3-Diynes are valuable building blocks for biologically active molecules and precursors of polymers, macrocycles and supramolecular structures. The combination of metal catalysts and molecular oxygen is considered a suitable protocol for the direct homocoupling of alkynes leading to diynes. Regarding the catalyst, palladium species have proved to be efficient and selective, but copper salts as co-catalysts and/or stoichiometric amounts of reoxidants are often needed. Palladium pincer complexes are presented as alternative palladium sources for the homocoupling of alkynes in an environmentally friendly solvent as polyethylene glycol, in the absence of any other co-catalyst and under aerobic conditions

The preparation of carboxylic acids from arylacetylenes has traditionally been carried out by ozonolysis or other harmful oxidants, alone or in presence of some metal catalysts. In spite of the safety, availability and environmental benignity of molecular oxygen, the selective cleavage of unsaturated hydrocarbons mediated by the latter oxidant has been rarely reported. Herein we wish to present the efficient preparation of carboxylic acid derivatives  from alkynes employing molecular oxygen as the only oxidant and in the presence of a Ni(II) salt and 1,2,4-triazole ligands

The distribution of the orientation genes in chromosomes is not random and may have function implications. In this work, we used complex networks to study the orientation of genes. We used as a case of study the 14 chromosomes of Plasmodium falciparum. We constructed the respective 14 networks with an average of 383 nodes (genes) and 1314 links (pairs of gene with inverse orientation). Node centralities of these nodes were used to study the structure of the network.

The objective of this research was to accelerate the composting process of solid waste generated in the agroindustry of the Amazon by inoculating composting beds with a solution of isolated native microorganisms on the surface of the CIPCA forests. The microorganisms identified in this work 2 fungi Aspergillus fumigatus, Penicillum sp. And the bacteria Bacillus subtillis and Pseudomos fluorences. A biomass of microorganisms, with a concentration of 1 X 10 ⁷ CFU*mL^-1 per isolated microorganism, was applied and sprayed 4 L*m^-1 3 of substrate to compost. The following variables were evaluated: temperature, humidity, pH, C/N texture and physical structure, organic matter, electrical conductivity and cation exchange capacity. The results indicated that the beds inoculated with the microbial solution reached the physical, chemical and biological characteristics of a mature compost with the difference of five weeks before the control bed. The response in these characteristics indicated that the inoculum solution significantly accelerates the composting process.