Ollila, S. O. H. | bioRxiv Science

bioRxiv2026

ActSeekN: A Structural-Motif-Based Pipeline for Interpretable Enzyme Function Annotation

Accurate enzyme function annotation remains a major bottleneck in genome analysis despite the rapid expansion of available protein sequence and structure data. Most existing methods rely on sequence similarity or machine-learning representations, which often perform poorly for proteins with low sequence identity or convergent evolutionary histories. Because enzymatic activity is determined by the three-dimensional arrangement of catalytic and binding-site residues, structure-based approaches offer a mechanistically grounded alternative. However, their broader application has been constrained by the limited size and coverage of curated active-site reference databases. To address this challenge, we developed ActSeekN, a structural-motif-based functional annotation pipeline that combines the ActSeek active-site search algorithm with a newly constructed large-scale reference database derived from AlphaFold-predicted structures, UniProt annotations, and curated catalytic residue information. This framework enables rapid and scalable identification of conserved catalytic motifs across structurally related proteins, allowing function to be transferred on the basis of local three-dimensional catalytic geometry rather than global sequence similarity. In this way, ActSeekN overcomes a central limitation of previous structure-based methods by expanding the searchable space of catalytic motifs while retaining mechanistic interpretability. Benchmarking against state-of-the-art machine-learning approaches demonstrates competitive or superior performance. Applications to yeast, human, and Trichoderma reesei proteomes refine existing annotations, complete partial EC assignments, and identify previously unrecognized enzymatic functions, highlighting ActSeekN as a powerful tool for genome annotation and biotechnology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/720574v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@f5da16org.highwire.dtl.DTLVardef@c0faa4org.highwire.dtl.DTLVardef@18765fforg.highwire.dtl.DTLVardef@3960b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Exploring Peptide-Based Nanodiscs Structure and Dynamics through Synergistic Approach of NMR Spectroscopy, SAS and MD Simulations

Peptide nanodiscs are promising anti-atherosclerosis therapeutics, drug delivery particles and structural biology tools. However, the lack of experimental methods for structural and dynamical characterization of these particles hinders their further development. Here we integrated nuclear magnetic resonance (NMR), small-angle x-ray scattering, and small-angle neutron scattering experiments with molecular dynamics (MD) simulations to investigate the structure and dynamics of peptide nanodiscs stabilized by the apolipoprotein A-I mimetic peptide 22A with therapeutic activity against atherosclerosis. This multi-technique approach takes advantage of combining average size and shape information from small-angle scattering, peptide site-specific information from NMR spectroscopy, and interpretative power of MD simulations. Our results reveal the intrinsic polydispersity in size of peptide nanodiscs, highlighting the importance of careful interpretation when using averaged experimental parameters. Our consensus model suggests that 22A peptides are predominantly in -helical configuration with a disordered inter-helical orientation around the lipid matrix. The terminal regions of the peptides display greater flexibility relative to the peptide core and an enhanced C-terminal exposure to solvent, which could facilitate interaction with the enzyme LCAT. Interestingly, our results indicate that peptides and lipids rotate together as a rigid body. The methodological approach described in this paper paves the way for the design of more stable and effective therapeutic nanodiscs and for the characterization of other biomolecular aggregates that are beyond the scope of current structural biology techniques. Significance StatementNanodiscs stabilized by 22A apoA-I mimetic peptides hold significant pharmaceutical potential for treating cardiovascular diseases by mimicking HDL functions, yet their development is hindered by the difficulty of characterizing disordered biomolecular systems. Standard structural biology techniques cannot readily resolve the structure and dynamics of these peptide nanodiscs, which is essential for rational therapeutic design. Here, we integrate complementary biophysical experiments with MD simulations to establish a consensus model of 22A peptide nanodisc structure, dynamics, and interactions with biological partners at molecular resolution. Beyond advancing peptide nanodisc design, our integrative methodology provides a generalizable framework for characterizing other disordered biomolecular assemblies that are similarly challenging to conventional structural approaches.

biochemistry↗