Enhancing efficiency of protein language models with minimal wet-lab data through few-shot learning.

Accurately modeling the protein fitness landscapes holds great importance for protein engineering. Pre-trained protein language models have achieved state-of-the-art performance in predicting protein fitness without wet-lab experimental data, but their accuracy and interpretability remain limited. On the other hand, traditional supervised deep learning models require abundant labeled training examples for performance improvements, posing a practical barrier. In this work, we introduce FSFP, a training strategy that can effectively optimize protein language models under extreme data scarcity for fitness prediction. By combining meta-transfer learning, learning to rank, and parameter-efficient fine-tuning, FSFP can significantly boost the performance of various protein language models using merely tens of labeled single-site mutants from the target protein. In silico benchmarks across 87 deep mutational scanning datasets demonstrate FSFP's superiority over both unsupervised and supervised baselines. Furthermore, we successfully apply FSFP to engineer the Phi29 DNA polymerase through wet-lab experiments, achieving a 25% increase in the positive rate. These results underscore the potential of our approach in aiding AI-guided protein engineering.

Detection and Analysis of Short Linear Motif-Based Protein-Protein Interactions with SLiMAn2 Web Server.

Interactomics is bringing a deluge of data regarding protein-protein interactions (PPIs) which are involved in various molecular processes in all types of cells. However, this information does not easily translate into direct and precise molecular interfaces. This limits our understanding of each interaction network and prevents their efficient modulation. A lot of the detected interactions involve recognition of short linear motifs (SLiMs) by a folded domain while others rely on domain-domain interactions. Functional SLiMs hide among a lot of spurious ones, making deeper analysis of interactomes tedious. Hence, actual contacts and direct interactions are difficult to identify.Consequently, there is a need for user-friendly bioinformatic tools, enabling rapid molecular and structural analysis of SLiM-based PPIs in a protein network. In this chapter, we describe the use of the new webserver SLiMAn to help digging into SLiM-based PPIs in an interactive fashion.

Analysis and Visualization of Protein Channels, Tunnels, and Pores with MOLEonline and ChannelsDB 2.0.

Channels, tunnels, and pores serve as pathways for the transport of molecules and ions through protein structures, thus participating to their functions. MOLEonline ( https://mole.upol.cz ) is an interactive web-based tool with enhanced capabilities for detecting and characterizing channels, tunnels, and pores within protein structures. MOLEonline has two distinct calculation modes for analysis of channel and tunnels or transmembrane pores. This application gives researchers rich analytical insights into channel detection, structural characterization, and physicochemical properties. ChannelsDB 2.0 ( https://channelsdb2.biodata.ceitec.cz/ ) is a comprehensive database that offers information on the location, geometry, and physicochemical characteristics of tunnels and pores within macromolecular structures deposited in Protein Data Bank and AlphaFill databases. These tunnels are sourced from manual deposition from literature and automatic detection using software tools MOLE and CAVER. MOLEonline and ChannelsDB visualization is powered by the LiteMol Viewer and Mol* viewer, ensuring a user-friendly workspace. This chapter provides an overview of user applications and usage.

Approaches to Backbone Flexibility in Protein-Protein Docking.

Proteins are the fundamental organic macromolecules in living systems that play a key role in a variety of biological functions including immunological detection, intracellular trafficking, and signal transduction. The docking of proteins has greatly advanced during recent decades and has become a crucial complement to experimental methods. Protein-protein docking is a helpful method for simulating protein complexes whose structures have not yet been solved experimentally. This chapter focuses on major search tactics along with various docking programs used in protein-protein docking algorithms, which include: direct search, exhaustive global search, local shape feature matching, randomized search, and broad category of post-docking approaches. As backbone flexibility predictions and interactions in high-resolution protein-protein docking remain important issues in the overall optimization context, we have put forward several methods and solutions used to handle backbone flexibility. In addition, various docking methods that are utilized for flexible backbone docking, including ATTRACT, FlexDock, FLIPDock, HADDOCK, RosettaDock, FiberDock, etc., along with their scoring functions, algorithms, advantages, and limitations are discussed. Moreover, what progress in search technology is expected, including not only the creation of new search algorithms but also the enhancement of existing ones, has been debated. As conformational flexibility is one of the most crucial factors affecting docking success, more work should be put into evaluating the conformational flexibility upon binding for a particular case in addition to developing new algorithms to replace the rigid body docking and scoring approach.

Sampling and Scoring in Protein-Protein Docking.

Protein-protein docking is considered one of the most important techniques supporting experimental proteomics. Recent developments in the field of computer science helped to improve this computational technique so that it better handles the complexity of protein nature. Sampling algorithms are responsible for the generation of numerous protein-protein ensembles. Unfortunately, a primary docking output comprises a set of both near-native poses and decoys. Application of the efficient scoring function helps to differentiate poses with the most favorable properties from those that are very unlikely to represent a natural state of the complex. This chapter explains the importance of sampling and scoring in the process of protein-protein docking. Moreover, it summarizes advances in the field.

From Multiple Protein Docking to Protein-Protein Docking at Interactome Level.

Molecular docking is used to anticipate the optimal orientation of a particular molecule to a target to form a stable complex. It makes predictions about the 3D structure of any complex based on the binding characteristics of the ligand and the target receptor usually a protein. It is an exceptionally useful tool, which is used as a model to study how ligands attach to proteins. Docking can also be used for studying the interaction of ligands and proteins to analyze inhibitory efficacy. The ligand may also be a protein, making it possible to study interactions between two different proteins using the numerous docking tools available for basic research on protein interactions. The protein-protein docking is a crucial approach to understanding the protein interactions and predicting the structure of protein complexes that have not yet been experimentally determined. Moreover, the protein-protein interactions can predict the function of target proteins and the drug-like properties of molecules. Therefore, protein docking assists in uncovering insights into protein interactions and also aids in a better understanding of molecular pathways/mechanisms. This chapter comprehends the various tools for protein-protein docking (pairwise and multiple), including their methodologies and analysis of output as results.

Docking Foundations: From Rigid to Flexible Docking.

Despite the development of methods for the experimental determination of protein structures, the dissonance between the number of known sequences and their solved structures is still enormous. This is particularly evident in protein-protein complexes. To fill this gap, diverse technologies have been developed to study protein-protein interactions (PPIs) in a cellular context including a range of biological and computational methods. The latter derive from techniques originally published and applied almost half a century ago and are based on interdisciplinary knowledge from the nexus of the fields of biology, chemistry, and physics about protein sequences, structures, and their folding. Protein-protein docking, the main protagonist of this chapter, is routinely treated as an integral part of protein research. Herein, we describe the basic foundations of the whole process in general terms, but step by step from protein representations through docking methods and evaluation of complexes to their final validation.

Software and Databases for Protein-Protein Docking.

Protein-protein interactions (PPIs) provide valuable insights for understanding the principles of biological systems and for elucidating causes of incurable diseases. One of the techniques used for computational prediction of PPIs is protein-protein docking calculations, and a variety of software has been developed. This chapter is a summary of software and databases used for protein-protein docking.

Machine Learning Methods in Protein-Protein Docking.

An exponential increase in the number of publications that address artificial intelligence (AI) usage in life sciences has been noticed in recent years, while new modeling techniques are constantly being reported. The potential of these methods is vast-from understanding fundamental cellular processes to discovering new drugs and breakthrough therapies. Computational studies of protein-protein interactions, crucial for understanding the operation of biological systems, are no exception in this field. However, despite the rapid development of technology and the progress in developing new approaches, many aspects remain challenging to solve, such as predicting conformational changes in proteins, or more "trivial" issues as high-quality data in huge quantities.Therefore, this chapter focuses on a short introduction to various AI approaches to study protein-protein interactions, followed by a description of the most up-to-date algorithms and programs used for this purpose. Yet, given the considerable pace of development in this hot area of computational science, at the time you read this chapter, the development of the algorithms described, or the emergence of new (and better) ones should come as no surprise.

Free-Docking and Template-Based Docking: Physics Versus Knowledge-Based Docking.

Docking methods can be used to predict the orientations of two or more molecules with respect of each other using a plethora of various algorithms, which can be based on the physics of interactions or can use information from databases and templates. The usability of these approaches depends on the type and size of the molecules, whose relative orientation will be estimated. The two most important limitations are (i) the computational cost of the prediction and (ii) the availability of the structural information for similar complexes. In general, if there is enough information about similar systems, knowledge-based and template-based methods can significantly reduce the computational cost while providing high accuracy of the prediction. However, if the information about the system topology and interactions between its partners is scarce, physics-based methods are more reliable or even the only choice. In this chapter, knowledge-, template-, and physics-based methods will be compared and briefly discussed providing examples of their usability with a special emphasis on physics-based protein-protein, protein-peptide, and protein-fullerene docking in the UNRES coarse-grained model.

Software for Predicting Binding Free Energy of Protein-Protein Complexes and Their Mutants.

Protein-protein binding affinity prediction is important for understanding complex biochemical pathways and to uncover protein interaction networks. Quantitative estimation of the binding affinity changes caused by mutations can provide critical information for protein function annotation and genetic disease diagnoses. The binding free energies of protein-protein complexes can be predicted using several computational tools. This chapter is a summary of software developed for the prediction of binding free energies for protein-protein complexes and their mutants.

Molecular Dynamics Simulations in Protein-Protein Docking.

Concerted interactions between all the cell components form the basis of biological processes. Protein-protein interactions (PPIs) constitute a tremendous part of this interaction network. Deeper insight into PPIs can help us better understand numerous diseases and lead to the development of new diagnostic and therapeutic strategies. PPI interfaces, until recently, were considered undruggable. However, it is now believed that the interfaces contain "hot spots," which could be targeted by small molecules. Such a strategy would require high-quality structural data of PPIs, which are difficult to obtain experimentally. Therefore, in silico modeling can complement or be an alternative to in vitro approaches. There are several computational methods for analyzing the structural data of the binding partners and modeling of the protein-protein dimer/oligomer structure. The major problem with in silico structure prediction of protein assemblies is obtaining sufficient sampling of protein dynamics. One of the methods that can take protein flexibility and the effects of the environment into account is Molecular Dynamics (MD). While sampling of the whole protein-protein association process with plain MD would be computationally expensive, there are several strategies to harness the method to PPI studies while maintaining reasonable use of resources. This chapter reviews known applications of MD in the PPI investigation workflows.

Computational Methods to Target Protein-Protein Interactions.

The chapter emphasizes the importance of understanding protein-protein interactions in cellular mechanisms and highlights the role of computational modeling in predicting these interactions. It discusses sequence-based approaches such as evolutionary trace (ET), correlated mutation analysis (CMA), and subtractive correlated mutation (SCM) for identifying crucial amino acid residues, considering interface conservation or evolutionary changes. The chapter also explores methods like differential ET, hidden-site class model, and spatial cluster detection (SCD) for interface specificity and spatial clustering. Furthermore, it examines approaches combining structural and sequential methodologies and evaluates modeled predictions through initiatives like critical assessment of prediction of interactions (CAPRI). Additionally, the chapter provides an overview of various software programs used for molecular docking, detailing their search, sampling, refinement and scoring stages, along with innovative techniques and tools like normal mode analysis (NMA) and adaptive Poisson-Boltzmann solver (APBS) for electrostatic calculations. These computational and experimental approaches are crucial for unraveling protein-protein interactions and aid in developing potential therapeutics for various diseases.

Assessment of Protein-Protein Docking Models Using Deep Learning.

Protein-protein interactions are involved in almost all processes in a living cell and determine the biological functions of proteins. To obtain mechanistic understandings of protein-protein interactions, the tertiary structures of protein complexes have been determined by biophysical experimental methods, such as X-ray crystallography and cryogenic electron microscopy. However, as experimental methods are costly in resources, many computational methods have been developed that model protein complex structures. One of the difficulties in computational protein complex modeling (protein docking) is to select the most accurate models among many models that are usually generated by a docking method. This article reviews advances in protein docking model assessment methods, focusing on recent developments that apply deep learning to several network architectures.

MDFit: automated molecular simulations workflow enables high throughput assessment of ligands-protein dynamics.

Molecular dynamics (MD) simulation is a powerful tool for characterizing ligand-protein conformational dynamics and offers significant advantages over docking and other rigid structure-based computational methods. However, setting up, running, and analyzing MD simulations continues to be a multi-step process making it cumbersome to assess a library of ligands in a protein binding pocket using MD. We present an automated workflow that streamlines setting up, running, and analyzing Desmond MD simulations for protein-ligand complexes using machine learning (ML) models. The workflow takes a library of pre-docked ligands and a prepared protein structure as input, sets up and runs MD with each protein-ligand complex, and generates simulation fingerprints for each ligand. Simulation fingerprints (SimFP) capture protein-ligand compatibility, including stability of different ligand-pocket interactions and other useful metrics that enable easy rank-ordering of the ligand library for pocket optimization. SimFPs from a ligand library are used to build & deploy ML models that predict binding assay outcomes and automatically infer important interactions. Unlike relative free-energy methods that are constrained to assess ligands with high chemical similarity, ML models based on SimFPs can accommodate diverse ligand sets. We present two case studies on how SimFP helps delineate structure-activity relationship (SAR) trends and explain potency differences across matched-molecular pairs of (1) cyclic peptides targeting PD-L1 and (2) small molecule inhibitors targeting CDK9.

[Advances in using artificial intelligence for predicting protein-ligand binding affinity].

The binding of proteins and ligands is a crucial aspect of life processes. The calculation of the protein-ligand binding affinity (PLBA) offers valuable insights into protein function, drug screening targets protein receptors, and enzyme modifications. In recent years, artificial intelligence (AI) has experienced rapid advancements, becoming widely used in PLBA prediction. This is attributed to its robust feature extraction ability, superior algorithm accuracy, and speedy calculations. Our paper aims to provide a comprehensive overview of AI predication process, associated resources, application scenarios, challenges, and potential solutions, serving as a valuable reference for the relevant research endeavors.

[Advances in bioinformatics-based protein function prediction].

With the increasing of computer power and rapid expansion of biological data, the application of bioinformatics tools has become the mainstream approach to address biological problems. The accurate identification of protein function by bioinformatics tools is crucial for both biomedical research and drug discovery, making it a hot topic of research. In this paper, we categorize bioinformatics-based protein function prediction methods into three categories: protein sequence-based methods, protein structure-based methods, and protein interaction networks-based methods. We further analyze these specific algorithms, highlighting the latest research advancements and providing valuable references for the application of bioinformatics-based protein function prediction in biomedical research and drug discovery.

MetalinksDB: a flexible and contextualizable resource of metabolite-protein interactions.

From the catalytic breakdown of nutrients to signaling, interactions between metabolites and proteins play an essential role in cellular function. An important case is cell-cell communication, where metabolites, secreted into the microenvironment, initiate signaling cascades by binding to intra- or extracellular receptors of neighboring cells. Protein-protein cell-cell communication interactions are routinely predicted from transcriptomic data. However, inferring metabolite-mediated intercellular signaling remains challenging, partially due to the limited size of intercellular prior knowledge resources focused on metabolites. Here, we leverage knowledge-graph infrastructure to integrate generalistic metabolite-protein with curated metabolite-receptor resources to create MetalinksDB. MetalinksDB is an order of magnitude larger than existing metabolite-receptor resources and can be tailored to specific biological contexts, such as diseases, pathways, or tissue/cellular locations. We demonstrate MetalinksDB's utility in identifying deregulated processes in renal cancer using multi-omics bulk data. Furthermore, we infer metabolite-driven intercellular signaling in acute kidney injury using spatial transcriptomics data. MetalinksDB is a comprehensive and customizable database of intercellular metabolite-protein interactions, accessible via a web interface (https://metalinks.omnipathdb.org/) and programmatically as a knowledge graph (https://github.com/biocypher/metalinks). We anticipate that by enabling diverse analyses tailored to specific biological contexts, MetalinksDB will facilitate the discovery of disease-relevant metabolite-mediated intercellular signaling processes.

A large-scale assessment of sequence database search tools for homology-based protein function prediction.

Sequence database searches followed by homology-based function transfer form one of the oldest and most popular approaches for predicting protein functions, such as Gene Ontology (GO) terms. These searches are also a critical component in most state-of-the-art machine learning and deep learning-based protein function predictors. Although sequence search tools are the basis of homology-based protein function prediction, previous studies have scarcely explored how to select the optimal sequence search tools and configure their parameters to achieve the best function prediction. In this paper, we evaluate the effect of using different options from among popular search tools, as well as the impacts of search parameters, on protein function prediction. When predicting GO terms on a large benchmark dataset, we found that BLASTp and MMseqs2 consistently exceed the performance of other tools, including DIAMOND-one of the most popular tools for function prediction-under default search parameters. However, with the correct parameter settings, DIAMOND can perform comparably to BLASTp and MMseqs2 in function prediction. Additionally, we developed a new scoring function to derive GO prediction from homologous hits that consistently outperform previously proposed scoring functions. These findings enable the improvement of almost all protein function prediction algorithms with a few easily implementable changes in their sequence homolog-based component. This study emphasizes the critical role of search parameter settings in homology-based function transfer and should have an important contribution to the development of future protein function prediction algorithms.

A curated rotamer library for common post-translational modifications of proteins.

MOTIVATION: Sidechain rotamer libraries of the common amino acids of a protein are useful for folded protein structure determination and for generating ensembles of intrinsically disordered proteins (IDPs). However, much of protein function is modulated beyond the translated sequence through the introduction of post-translational modifications (PTMs). RESULTS: In this work, we have provided a curated set of side chain rotamers for the most common PTMs derived from the RCSB PDB database, including phosphorylated, methylated, and acetylated sidechains. Our rotamer libraries improve upon existing methods such as SIDEpro, Rosetta, and AlphaFold3 in predicting the experimental structures for PTMs in folded proteins. In addition, we showcase our PTM libraries in full use by generating ensembles with the Monte Carlo Side Chain Entropy (MCSCE) for folded proteins, and combining MCSCE with the Local Disordered Region Sampling algorithms within IDPConformerGenerator for proteins with intrinsically disordered regions. AVAILABILITY AND IMPLEMENTATION: The codes for dihedral angle computations and library creation are available at https://github.com/THGLab/ptm_sc.git.