Gating movements and ion permeation in HCN4 pacemaker channels.

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

Anisotropic Network Analysis of Open/Closed HCN4 Channel Advocates Asymmetric Subunit Cooperativity in cAMP Modulation of Gating.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are opened in an allosteric manner by membrane hyperpolarization and cyclic nucleotides such as cAMP. Because of conflicting reports from experimental studies on whether cAMP binding to the four available binding sites in the channel tetramer operates cooperatively in gating, we employ here a computational approach as a promising route to examine ligand-induced conformational changes after binding to individual sites. By combining an elastic network model (ENM) with linear response theory (LRT) for modeling the apo-holo transition of the cyclic nucleotide-binding domain (CNBD) in HCN channels, we observe a distinct pattern of cooperativity matching the "positive-negative-positive" cooperativity reported from functional studies. This cooperativity pattern is highly conserved among HCN subtypes (HCN4, HCN1), but only to a lesser extent visible in structurally related channels, which are only gated by voltage (KAT1) or cyclic nucleotides (TAX4). This suggests an inherent cooperativity between subunits in HCN channels as part of a ligand-triggered gating mechanism in these channels.

Biotite: new tools for a versatile Python bioinformatics library.

BACKGROUND: Biotite is a program library for sequence and structural bioinformatics written for the Python programming language. It implements widely used computational methods into a consistent and accessible package. This allows for easy combination of various data analysis, modeling and simulation methods. RESULTS: This article presents major functionalities introduced into Biotite since its original publication. The fields of application are shown using concrete examples. We show that the computational performance of Biotite for bioinformatics tasks is comparable to individual, special purpose software systems specifically developed for the respective single task. CONCLUSIONS: The results show that Biotite can be used as program library to either answer specific bioinformatics questions and simultaneously allow the user to write entire, self-contained software applications with sufficient performance for general application.

Mainzelliste SecureEpiLinker (MainSEL): privacy-preserving record linkage using secure multi-party computation.

MOTIVATION: Record Linkage has versatile applications in real-world data analysis contexts, where several datasets need to be linked on the record level in the absence of any exact identifier connecting related records. An example are medical databases of patients, spread across institutions, that have to be linked on personally identifiable entries like name, date of birth or ZIP code. At the same time, privacy laws may prohibit the exchange of this personally identifiable information (PII) across institutional boundaries, ruling out the outsourcing of the record linkage task to a trusted third party. We propose to employ privacy-preserving record linkage (PPRL) techniques that prevent, to various degrees, the leakage of PII while still allowing for the linkage of related records. RESULTS: We develop a framework for fault-tolerant PPRL using secure multi-party computation with the medical record keeping software Mainzelliste as the data source. Our solution does not rely on any trusted third party and all PII is guaranteed to not leak under common cryptographic security assumptions. Benchmarks show the feasibility of our approach in realistic networking settings: linkage of a patient record against a database of 10 000 records can be done in 48 s over a heavily delayed (100 ms) network connection, or 3.9 s with a low-latency connection. AVAILABILITY AND IMPLEMENTATION: The source code of the sMPC node is freely available on Github at https://github.com/medicalinformatics/SecureEpilinker subject to the AGPLv3 license. The source code of the modified Mainzelliste is available at https://github.com/medicalinformatics/MainzellisteSEL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

EasySMPC: a simple but powerful no-code tool for practical secure multiparty computation.

BACKGROUND: Modern biomedical research is data-driven and relies heavily on the re-use and sharing of data. Biomedical data, however, is subject to strict data protection requirements. Due to the complexity of the data required and the scale of data use, obtaining informed consent is often infeasible. Other methods, such as anonymization or federation, in turn have their own limitations. Secure multi-party computation (SMPC) is a cryptographic technology for distributed calculations, which brings formally provable security and privacy guarantees and can be used to implement a wide-range of analytical approaches. As a relatively new technology, SMPC is still rarely used in real-world biomedical data sharing activities due to several barriers, including its technical complexity and lack of usability. RESULTS: To overcome these barriers, we have developed the tool EasySMPC, which is implemented in Java as a cross-platform, stand-alone desktop application provided as open-source software. The tool makes use of the SMPC method Arithmetic Secret Sharing, which allows to securely sum up pre-defined sets of variables among different parties in two rounds of communication (input sharing and output reconstruction) and integrates this method into a graphical user interface. No additional software services need to be set up or configured, as EasySMPC uses the most widespread digital communication channel available: e-mails. No cryptographic keys need to be exchanged between the parties and e-mails are exchanged automatically by the software. To demonstrate the practicability of our solution, we evaluated its performance in a wide range of data sharing scenarios. The results of our evaluation show that our approach is scalable (summing up 10,000 variables between 20 parties takes less than 300 s) and that the number of participants is the essential factor. CONCLUSIONS: We have developed an easy-to-use "no-code solution" for performing secure joint calculations on biomedical data using SMPC protocols, which is suitable for use by scientists without IT expertise and which has no special infrastructure requirements. We believe that innovative approaches to data sharing with SMPC are needed to foster the translation of complex protocols into practice.

Record linkage based patient intersection cardinality for rare disease studies using Mainzelliste and secure multi-party computation.

BACKGROUND: The low number of patients suffering from any given rare diseases poses a difficult problem for medical research: With the exception of some specialized biobanks and disease registries, potential study participants' information are disjoint and distributed over many medical institutions. Whenever some of those facilities are in close proximity, a significant overlap of patients can reasonably be expected, further complicating statistical study feasibility assessments and data gathering. Due to the sensitive nature of medical records and identifying data, data transfer and joint computations are often forbidden by law or associated with prohibitive amounts of effort. To alleviate this problem and to support rare disease research, we developed the Mainzelliste Secure EpiLinker (MainSEL) record linkage framework, a secure Multi-Party Computation based application using trusted-third-party-less cryptographic protocols to perform privacy-preserving record linkage with high security guarantees. In this work, we extend MainSEL to allow the record linkage based calculation of the number of common patients between institutions. This allows privacy-preserving statistical feasibility estimations for further analyses and data consolidation. Additionally, we created easy to deploy software packages using microservice containerization and continuous deployment/continuous integration. We performed tests with medical researchers using MainSEL in real-world medical IT environments, using synthetic patient data. RESULTS: We show that MainSEL achieves practical runtimes, performing 10 000 comparisons in approximately 5 minutes. Our approach proved to be feasible in a wide range of network settings and use cases. The "lessons learned" from the real-world testing show the need to explicitly support and document the usage and deployment for both analysis pipeline integration and researcher driven ad-hoc analysis use cases, thus clarifying the wide applicability of our software. MainSEL is freely available under: https://github.com/medicalinformatics/MainSEL CONCLUSIONS: MainSEL performs well in real-world settings and is a useful tool not only for rare disease research, but medical research in general. It achieves practical runtimes, improved security guarantees compared to existing solutions, and is simple to deploy in strict clinical IT environments. Based on the "lessons learned" from the real-word testing, we hope to enable a wide range of medical researchers to meet their needs and requirements using modern privacy-preserving technologies.

SPIKE: secure and private investigation of the kidney exchange problem.

BACKGROUND: The kidney exchange problem (KEP) addresses the matching of patients in need for a replacement organ with compatible living donors. Ideally many medical institutions should participate in a matching program to increase the chance for successful matches. However, to fulfill legal requirements current systems use complicated policy-based data protection mechanisms that effectively exclude smaller medical facilities to participate. Employing secure multi-party computation (MPC) techniques provides a technical way to satisfy data protection requirements for highly sensitive personal health information while simultaneously reducing the regulatory burdens. RESULTS: We have designed, implemented, and benchmarked SPIKE, a secure MPC-based privacy-preserving KEP protocol which computes a locally optimal solution by finding matching donor-recipient pairs in a graph structure. SPIKE matches 40 pairs in cycles of length 2 in less than 4 min and outperforms the previous state-of-the-art protocol by a factor of [Formula: see text] in runtime while providing medically more robust solutions. CONCLUSIONS: We show how to solve the KEP in a robust and privacy-preserving manner achieving significantly more practical performance than the current state-of-the-art (Breuer et al., WPES'20 and CODASPY'22). The usage of MPC techniques fulfills many data protection requirements on a technical level, allowing smaller health care providers to directly participate in a kidney exchange with reduced legal processes. As sensitive data are not leaving the institutions' network boundaries, the patient data underlie a higher level of protection than in the currently employed (centralized) systems. Furthermore, due to reduced legal barriers, the proposed decentralized system might be simpler to implement in a transnational, intereuropean setting with mixed (national) data protecion laws.

Consistent Quantification of Complex Dynamics via a Novel Statistical Complexity Measure.

Natural systems often show complex dynamics. The quantification of such complex dynamics is an important step in, e.g., characterization and classification of different systems or to investigate the effect of an external perturbation on the dynamics. Promising routes were followed in the past using concepts based on (Shannon's) entropy. Here, we propose a new, conceptually sound measure that can be pragmatically computed, in contrast to pure theoretical concepts based on, e.g., Kolmogorov complexity. We illustrate the applicability using a toy example with a control parameter and go on to the molecular evolution of the HIV1 protease for which drug treatment can be regarded as an external perturbation that changes the complexity of its molecular evolutionary dynamics. In fact, our method identifies exactly those residues which are known to bind the drug molecules by their noticeable signal. We furthermore apply our method in a completely different domain, namely foreign exchange rates, and find convincing results as well.

Inferring functional units in ion channel pores via relative entropy.

Coarse-grained protein models approximate the first-principle physical potentials. Among those modeling approaches, the relative entropy framework yields promising and physically sound results, in which a mapping from the target protein structure and dynamics to a model is defined and subsequently adjusted by an entropy minimization of the model parameters. Minimization of the relative entropy is equivalent to maximization of the likelihood of reproduction of (configurational ensemble) observations by the model. In this study, we extend the relative entropy minimization procedure beyond parameter fitting by a second optimization level, which identifies the optimal mapping to a (dimension-reduced) topology. We consider anisotropic network models of a diverse set of ion channels and assess our findings by comparison to experimental results.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Substitution matrix based color schemes for sequence alignment visualization.

BACKGROUND: Visualization of multiple sequence alignments often includes colored symbols, usually characters encoding amino acids, according to some (physical) properties, such as hydrophobicity or charge. Typically, color schemes are created manually, so that equal or similar colors are assigned to amino acids that share similar properties. However, this assessment is subjective and may not represent the similarity of symbols very well. RESULTS: In this article we propose a different approach for color scheme creation: We leverage the similarity information of a substitution matrix to derive an appropriate color scheme. Similar colors are assigned to high scoring pairs of symbols, distant colors are assigned to low scoring pairs. In order to find these optimal points in color space a simulated annealing algorithm is employed. CONCLUSIONS: Using the substitution matrix as basis for a color scheme is consistent with the alignment, which itself is based on the very substitution matrix. This approach allows fully automatic generation of new color schemes, even for special purposes which have not been covered, yet, including schemes for structural alphabets or schemes that are adapted for people with color vision deficiency.

Riboswitching with ciprofloxacin-development and characterization of a novel RNA regulator.

RNA molecules play important and diverse regulatory roles in the cell. Inspired by this natural versatility, RNA devices are increasingly important for many synthetic biology applications, e.g. optimizing engineered metabolic pathways, gene therapeutics or building up complex logical units. A major advantage of RNA is the possibility of de novo design of RNA-based sensing domains via an in vitro selection process (SELEX). Here, we describe development of a novel ciprofloxacin-responsive riboswitch by in vitro selection and next-generation sequencing-guided cellular screening. The riboswitch recognizes the small molecule drug ciprofloxacin with a KD in the low nanomolar range and adopts a pseudoknot fold stabilized by ligand binding. It efficiently interferes with gene expression both in lower and higher eukaryotes. By controlling an auxotrophy marker and a resistance gene, respectively, we demonstrate efficient, scalable and programmable control of cellular survival in yeast. The applied strategy for the development of the ciprofloxacin riboswitch is easily transferrable to any small molecule target of choice and will thus broaden the spectrum of RNA regulators considerably.

Mechanical transduction of cytoplasmic-to-transmembrane-domain movements in a hyperpolarization-activated cyclic nucleotide-gated cation channel.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels play a critical role in the control of pacemaking in the heart and repetitive firing in neurons. In HCN channels, the intracellular cyclic nucleotide-binding domain (CNBD) is connected to the transmembrane portion of the channel (TMPC) through a helical domain, the C-linker. Although this domain is critical for mechanical signal transduction, the conformational dynamics in the C-linker that transmit the nucleotide-binding signal to the HCN channel pore are unknown. Here, we use linear response theory to analyze conformational changes in the C-linker of the human HCN1 protein, which couple cAMP binding in the CNBD with gating in the TMPC. By applying a force to the tip of the so-called "elbow" of the C-linker, the coarse-grained calculations recapitulate the same conformational changes triggered by cAMP binding in experimental studies. Furthermore, in our simulations, a displacement of the C-linker parallel to the membrane plane (i.e. horizontally) induced a rotational movement resulting in a distinct tilting of the transmembrane helices. This movement, in turn, increased the distance between the voltage-sensing S4 domain and the surrounding transmembrane domains and led to a widening of the intracellular channel gate. In conclusion, our computational approach, combined with experimental data, thus provides a more detailed understanding of how cAMP binding is mechanically coupled over long distances to promote voltage-dependent opening of HCN channels.

hoDCA: higher order direct-coupling analysis.

BACKGROUND: Direct-coupling analysis (DCA) is a method for protein contact prediction from sequence information alone. Its underlying principle is parameter estimation for a Hamiltonian interaction function stemming from a maximum entropy model with one- and two-point interactions. Vastly growing sequence databases enable the construction of large multiple sequence alignments (MSA). Thus, enough data exists to include higher order terms, such as three-body correlations. RESULTS: We present an implementation of hoDCA, which is an extension of DCA by including three-body interactions into the inverse Ising problem posed by parameter estimation. In a previous study, these three-body-interactions improved contact prediction accuracy for the PSICOV benchmark dataset. Our implementation can be executed in parallel, which results in fast runtimes and makes it suitable for large-scale application. CONCLUSION: Our hoDCA software allows improved contact prediction using the Julia language, leveraging power of multi-core machines in an automated fashion.

Biotite: a unifying open source computational biology framework in Python.

BACKGROUND: As molecular biology is creating an increasing amount of sequence and structure data, the multitude of software to analyze this data is also rising. Most of the programs are made for a specific task, hence the user often needs to combine multiple programs in order to reach a goal. This can make the data processing unhandy, inflexible and even inefficient due to an overhead of read/write operations. Therefore, it is crucial to have a comprehensive, accessible and efficient computational biology framework in a scripting language to overcome these limitations. RESULTS: We have developed the Python package Biotite: a general computational biology framework, that represents sequence and structure data based on NumPyndarrays. Furthermore the package contains seamless interfaces to biological databases and external software. The source code is freely accessible at https://github.com/biotite-dev/biotite . CONCLUSIONS: Biotite is unifying in two ways: At first it bundles popular tasks in sequence analysis and structural bioinformatics in a consistently structured package. Secondly it adresses two groups of users: novice programmers get an easy access to Biotite due to its simplicity and the comprehensive documentation. On the other hand, advanced users can profit from its high performance and extensibility. They can implement their algorithms upon Biotite, so they can skip writing code for general functionality (like file parsers) and can focus on what their software makes unique.

StreaMD: Advanced analysis of molecular dynamics using R.

Gromacs is one of the most popular molecular simulation suites currently available. In this contribution we present streaMD, the first interface between Gromacs trajectory files and the statistical language R. The amount of data created due to ever increasing computational power renders fast and efficient analysis of trajectories into a challenge. Especially as standard approaches such as root-mean square fluctuations and the like provide only limited physical insight. In our streaMD package integration of the Gromacs I/O libraries with advanced, graph-based analysis methods as the java library Stream leads to both: improved speed and analysis depth. We benchmark our results and highlight the applicability of the package by an interesting problem in RNA design, namely the interaction of tetracycline with an aptamer. © 2018 Wiley Periodicals, Inc.

PFASUM: a substitution matrix from Pfam structural alignments.

BACKGROUND: Detecting homologous protein sequences and computing multiple sequence alignments (MSA) are fundamental tasks in molecular bioinformatics. These tasks usually require a substitution matrix for modeling evolutionary substitution events derived from a set of aligned sequences. Over the last years, the known sequence space increased drastically and several publications demonstrated that this can lead to significantly better performing matrices. Interestingly, matrices based on dated sequence datasets are still the de facto standard for both tasks even though their data basis may limit their capabilities. We address these aspects by presenting a new substitution matrix series called PFASUM. These matrices are derived from Pfam seed MSAs using a novel algorithm and thus build upon expert ground truth data covering a large and diverse sequence space. RESULTS: We show results for two use cases: First, we tested the homology search performance of PFASUM matrices on up-to-date ASTRAL databases with varying sequence similarity. Our study shows that the usage of PFASUM matrices can lead to significantly better homology search results when compared to conventional matrices. PFASUM matrices with comparable relative entropies to the commonly used substitution matrices BLOSUM50, BLOSUM62, PAM250, VTML160 and VTML200 outperformed their corresponding counterparts in 93% of all test cases. A general assessment also comparing matrices with different relative entropies showed that PFASUM matrices delivered the best homology search performance in the test set. Second, our results demonstrate that the usage of PFASUM matrices for MSA construction improves their quality when compared to conventional matrices. On up-to-date MSA benchmarks, at least 60% of all MSAs were reconstructed in an equal or higher quality when using MUSCLE with PFASUM31, PFASUM43 and PFASUM60 matrices instead of conventional matrices. This rate even increases to at least 76% for MSAs containing similar sequences. CONCLUSIONS: We present the novel PFASUM substitution matrices derived from manually curated MSA ground truth data covering the currently known sequence space. Our results imply that PFASUM matrices improve homology search performance as well as MSA quality in many cases when compared to conventional substitution matrices. Hence, we encourage the usage of PFASUM matrices and especially PFASUM60 for these specific tasks.

Cleavage Product Accumulation Decreases the Activity of Cutinase during PET Hydrolysis.

The Fusarium solani cutinase (FsC) is a promising candidate for the enzymatic degradation of the synthetic polyester polyethylene terephthalate (PET) but still suffers from a lack of activity. Using atomic MD simulations with different concentrations of cleavage product ethylene glycol (EG), we show influences of EG on the dynamic of FsC. We observed accumulation of EG in the active site region reducing the local flexibility of FsC. Furthermore, we used a coarse-grained mechanical model to investigate whether substrate binding in the active site causes an induced fit. We observed this supposed induced fit or "breath-like" movement during substrate binding indicating that the active site has to be flexible for substrate conversion. This guides rational design: mutants with an increased flexibility near the active site should be considered to compensate the solvent-mediated reduction in activity.

Addressing inaccuracies in BLOSUM computation improves homology search performance.

BACKGROUND: BLOSUM matrices belong to the most commonly used substitution matrix series for protein homology search and sequence alignments since their publication in 1992. In 2008, Styczynski et al. discovered miscalculations in the clustering step of the matrix computation. Still, the RBLOSUM64 matrix based on the corrected BLOSUM code was reported to perform worse at a statistically significant level than the BLOSUM62. Here, we present a further correction of the (R)BLOSUM code and provide a thorough performance analysis of BLOSUM-, RBLOSUM- and the newly derived CorBLOSUM-type matrices. Thereby, we assess homology search performance of these matrix-types derived from three different BLOCKS databases on all versions of the ASTRAL20, ASTRAL40 and ASTRAL70 subsets resulting in 51 different benchmarks in total. Our analysis is focused on two of the most popular BLOSUM matrices - BLOSUM50 and BLOSUM62. RESULTS: Our study shows that fixing small errors in the BLOSUM code results in substantially different substitution matrices with a beneficial influence on homology search performance when compared to the original matrices. The CorBLOSUM matrices introduced here performed at least as good as their BLOSUM counterparts in ∼75 % of all test cases. On up-to-date ASTRAL databases BLOSUM matrices were even outperformed by CorBLOSUM matrices in more than 86 % of the times. In contrast to the study by Styczynski et al., the tested RBLOSUM matrices also outperformed the corresponding BLOSUM matrices in most of the cases. Comparing the CorBLOSUM with the RBLOSUM matrices revealed no general performance advantages for either on older ASTRAL releases. On up-to-date ASTRAL databases however CorBLOSUM matrices performed better than their RBLOSUM counterparts in ∼74 % of the test cases. CONCLUSIONS: Our results imply that CorBLOSUM type matrices outperform the BLOSUM matrices on a statistically significant level in most of the cases, especially on up-to-date databases such as ASTRAL ≥2.01. Additionally, CorBLOSUM matrices are closer to those originally intended by Henikoff and Henikoff on a conceptual level. Hence, we encourage the usage of CorBLOSUM over (R)BLOSUM matrices for the task of homology search.