Protonation-State-Dependent Communication in Cytochrome c Oxidase.

Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both a redox enzyme and a proton pump, proton transfer into the protein toward the BNC or toward a proton loading site (and ultimately through the membrane) must be highly regulated. The PR → F transition is the first step in a catalytic cycle that requires proton transfer from the bulk at the N-side to the BNC. Molecular dynamics simulations of the PR → F intermediate of this transition, with 16 different combinations of protonation states of key residues in the D- and K-channel, show the impact of the K-channel on the D-channel to be protonation-state dependent. Strength as well as means of communication, correlations in positions, or communication along the hydrogen-bonded network depends on the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 is regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286. In contrast, proton passage through the D-channel is hindered by N139's preference for a closed conformation in situations with protonated E286.

Structural and Vibrational Characterization of the Chromophore Binding Site of Bacterial Phytochrome Agp1.

Agp1 is a prototypical bacterial phytochrome from Agrobacterium fabrum harboring a biliverdin cofactor which reversibly photoconverts between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) states. The reaction mechanism involves the isomerization of the bilin-chromophore followed by large structural changes of the protein matrix that are coupled to protonation dynamics at the chromophore binding site. Histidines His250 and His280 participate in this process. Although the three-dimensional structure of Agp1 has been solved at high resolution, the precise position of hydrogen atoms and protonation pattern in the chromophore binding pocket has not been investigated yet. Here, we present protonated structure models of Agp1 in the Pr state involving appropriately placed hydrogen atoms that were generated by hybrid quantum mechanics/molecular mechanics- and electrostatic calculations and validated against experimental structural- and spectroscopic data. Although the effect of histidine protonation on the vibrational spectra is weak, our results favor charge neutral H250 and H280 both protonated at Nε. However, a neutral H250 with a proton at Nε and a cationic H280 may also be possible. Furthermore, the present QM/MM calculations of IR and Raman spectra of Agp1 containing isotope-labeled BV provide a detailed vibrational assignment of the biliverdin modes in the fingerprint region.

Redox induced protonation of heme propionates in cytochrome c oxidase: Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations.

Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3 have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a3 propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3 only protonation of one heme a3 propionates was observed. Our data supports the postulated scenario where the heme a3 propionates are involved in the proton pathway.

RETRACTED: Protonation State-Dependent Communication in Cytochrome c Oxidase.

Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The O→E intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286.

Proton transfer in the K-channel analog of B-type Cytochrome c oxidase from Thermus thermophilus.

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

Lysine 362 in cytochrome c oxidase regulates opening of the K-channel via changes in pKA and conformation.

The metabolism of aerobic life uses the conversion of molecular oxygen to water as an energy source. This reaction is catalyzed by cytochrome e oxidase (CeO) consuming four electrons and four protons, which move along specific routes. While all four electrons are transferred via the same cofactors to the binuclear reaction center (BNC), the protons take two different routes in the A-type CeO, i.e., two of the four chemical protons consumed in the reaction arrive via the D-channel in the oxidative first half starting after oxygen binding. The other two chemical protons enter via the K-channel in the reductive second half of the reaction cycle. To date, the mechanism behind these separate proton transport pathways has not been understood. In this study, we propose a model that can explain the reaction-step specific opening and closing of the K-channel by conformational and pKA changes of its central lysine 362. Molecular dynamics simulations reveal an upward movement of Lys362 towards the BNC, which had already been supposed by several experimental studies. Redox state-dependent pKA calculations provide evidence that Lys362 may protonate transiently, thereby opening the K-channel only in the reductive second half of the reaction cycle. From our results, we develop a model that assigns a key role to Lys362 in the proton gating between the two proton input channels of the A-type CeO.

Understanding selectin counter-receptor binding from electrostatic energy computations and experimental binding studies.

Higher organisms defend themselves against invading micro-organisms and harmful substances with their immune system. Key players of the immune system are the white blood cells (WBC), which in case of infection move in an extravasation process from blood vessels toward infected tissue promoting inflammation. This process starts with the attachment of the WBC to the blood vessel wall, mediated by protein pair interactions of selectins and counter-receptors (C-R). Individual selectin C-R binding is weak and varies only moderately between the three selectin types. Multivalency enhances such small differences, rendering selectin-binding type specific. In this work, we study selectin C-R binding, the initial step of extravasation. We performed electrostatic energy computations based on the crystal structure of one selectin type co-crystallized with the ligating part of the C-R. The agreement with measured free energies of binding is satisfactory. Additionally, we modeled selectin mutant structures in order to explain differences in binding of the different selectin types. To verify our modeling procedures, surface plasmon resonance data were measured for several mutants and compared with computed binding affinities. Binding affinities computed with soaked rather than co-crystallized selectin C-R structures do not agree with measured data. Hence, these structures are inappropriate to describe the binding mode. The analysis of selectin/C-R binding unravels the role played by individual molecular components in the binding event. This opens new avenues to prevent immune system malfunction, designing drugs that can control inflammatory processes by moderating selectin C-R binding.

Exploring the possible role of Glu286 in CcO by electrostatic energy computations combined with molecular dynamics.

Cytochrome c oxidase (CcO) is a central enzyme in aerobic life catalyzing the conversion of molecular oxygen to water and utilizing the chemical energy to pump protons and establish an electrochemical gradient. Despite intense research, it is not understood how CcO achieves unidirectional proton transport and avoids short circuiting the proton pump. Within this work, we analyzed the potential role of Glu286 as a proton valve. We performed unconstrained MD simulations of CcO with an explicit membrane for up to 80 ns. Those MD simulations revealed that deprotonated Glu286 (Glu286-) is repelled by the negatively charged propionic acid PRD of heme a3. Thus, it destabilizes a potential linear chain of waters in the hydrophobic cavity connecting Glu286 with PRD and the binuclear center (BNC). Conversely, protonated Glu286 (Glu286H) may remain in an upward position (oriented toward PRD) and can stabilize the connecting linear water chain in the hydrophobic cavity. We calculated the pKa of Glu286 under physiological conditions to be above 12, but this value decreases to about 9 under increased water accessibility of Glu286. The latter value is in accordance with experimental measurements. In the time course of MD simulation, we also observed conformations where Glu286 bridges between water molecules located on both sides (the D channel being connected to the N side and the hydrophobic cavity), which might lead to proton backflow.

VaccImm: simulating peptide vaccination in cancer therapy.

BACKGROUND: Despite progress in conventional cancer therapies, cancer is still one of the leading causes of death in industrial nations. Therefore, an urgent need of progress in fighting cancer remains. A promising alternative to conventional methods is immune therapy. This relies on the fact that low-immunogenic tumours can be eradicated if an immune response against them is induced. Peptide vaccination is carried out by injecting tumour peptides into a patient to trigger a specific immune response against the tumour in its entirety. However, peptide vaccination is a highly complicated treatment and currently many factors like the optimal number of epitopes are not known precisely. Therefore, it is necessary to evaluate how certain parameters influence the therapy. RESULTS: We present the VaccImm Server that allows users to simulate peptide vaccination in cancer therapy. It uses an agent-based model that simulates peptide vaccination by explicitly modelling the involved cells (immune system and cancer) as well as molecules (antibodies, antigens and semiochemicals). As a new feature, our model uses real amino acid sequences to represent molecular binding sites of relevant immune cells. The model is used to generate detailed statistics of the population sizes and states of the single cell types over time. This makes the VaccImm web server well suited to examine the parameter space of peptide vaccination in silico. VaccImm is publicly available without registration on the web at http://bioinformatics.charite.de/vaccimm; all major browsers are supported. CONCLUSIONS: The VaccImm Server provides a convenient way to analyze properties of peptide vaccination in cancer therapy. Using the server, we could gain interesting insights into peptide vaccination that reveal the complex and patient-specific nature of peptide vaccination.

Development of immune-specific interaction potentials and their application in the multi-agent-system VaccImm.

Peptide vaccination in cancer therapy is a promising alternative to conventional methods. However, the parameters for this personalized treatment are difficult to access experimentally. In this respect, in silico models can help to narrow down the parameter space or to explain certain phenomena at a systems level. Herein, we develop two empirical interaction potentials specific to B-cell and T-cell receptor complexes and validate their applicability in comparison to a more general potential. The interaction potentials are applied to the model VaccImm which simulates the immune response against solid tumors under peptide vaccination therapy. This multi-agent system is derived from another immune system simulator (C-ImmSim) and now includes a module that enables the amino acid sequence of immune receptors and their ligands to be taken into account. The multi-agent approach is combined with approved methods for prediction of major histocompatibility complex (MHC)-binding peptides and the newly developed interaction potentials. In the analysis, we critically assess the impact of the different modules on the simulation with VaccImm and how they influence each other. In addition, we explore the reasons for failures in inducing an immune response by examining the activation states of the immune cell populations in detail.In summary, the present work introduces immune-specific interaction potentials and their application to the agent-based model VaccImm which simulates peptide vaccination in cancer therapy.

Theoretical modeling techniques and their impact on tumor immunology.

Currently, cancer is one of the leading causes of death in industrial nations. While conventional cancer treatment usually results in the patient suffering from severe side effects, immunotherapy is a promising alternative. Nevertheless, some questions remain unanswered with regard to using immunotherapy to treat cancer hindering it from being widely established. To help rectify this deficit in knowledge, experimental data, accumulated from a huge number of different studies, can be integrated into theoretical models of the tumor-immune system interaction. Many complex mechanisms in immunology and oncology cannot be measured in experiments, but can be analyzed by mathematical simulations. Using theoretical modeling techniques, general principles of tumor-immune system interactions can be explored and clinical treatment schedules optimized to lower both tumor burden and side effects. In this paper, we aim to explain the main mathematical and computational modeling techniques used in tumor immunology to experimental researchers and clinicians. In addition, we review relevant published work and provide an overview of its impact to the field.