Parkinson's disease (PD), the second most common age-related neurodegenerative disorder worldwide, is associated with mutations in several genes, one of which is LRRK2 (leucine-rich repeat kinase 2). Despite cryo-EM structures elucidating the active and inactive architectures of the wild type (WT), the regulatory mechanisms and intermediate conformational states (INTs) that facilitate the transition between these forms, as well as the distinctions in the G2019S mutant, remain unclear. Here, we elucidated how mutations reshape the conformational dynamics through molecular dynamics simulations, highlighting the salt bridge contributions of the E1920-R2026 and K1906-D2017 residue pairs and fluctuations of the activation loop (A-loop) lid in stabilizing specific states. Furthermore, the reconstructed two-dimensional Well-Tempered Metadynamics (2D WT-MetaD) free-energy landscape along A-loop and K1906-E1920 coordinates revealed two distinct activation/inactivation pathways. Interestingly, the inactivation pathway in WT revealed a metastable "semi-open" state, while the G2019S mutation favored activation by decreasing the inactive-to-active energy barrier; however, once the active state was formed, it became comparatively stable and required more energy for the active-to-inactive transition than the WT. Thus, the G2019S mutation facilitated activation and remained in its active form, providing a mechanistic basis for its uncontrolled kinase hyperactivity. To reinforce the MetaD-derived stability of the G2019S active conformation over WT, we assessed the binding of the type-I inhibitor LRRK2-IN-1 for active state conformational rearrangements in the ligand-bound state. The absolute binding free-energy consistently showed that the ligand preferentially stabilizes the mutant active state, highlighting its binding affinity and modestly increased inhibitor potency, relative to WT. Finally, two-dimensional Umbrella Sampling (2D-US) showed apo-state transitions arising from DYGI (D2017-Y2018-G2019-I2020) rearrangements and αC-helix coupling via COM distances of K1906-E1920 and E1920-R2026 residue pairs. The results revealed that the mutant (G2019S) follows two routes with four INTs.

This chapter aims to provide the reader with an overview of how to leverage computational algorithms toward improving the affinity of an antibody for a particular target. In most cases, structural information is required; however, the prerequisite information is not always available. Here, we take a bottom-up approach that will guide readers through scenarios where data such as antibody apo or complex structures are unknown. The focus of the chapter is on how computational tools and workflows can be used to address these limitations when attempting to identify an affinity-matured antibody variant with desirable properties. We begin with an overview of antibody structure prediction, followed by predictions of antibody-antigen complexes, antibody affinity, stability, and developability. Finally, we conclude this chapter with guidance notes and best practices for antibody computational design.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated anion channel whose function is tightly linked to its conformational dynamics and is influenced by the composition of its membrane lipid environment. Despite high-resolution three-dimensional (3D) structures, the molecular determinants that stabilize specific CFTR conformations and enable ion conduction remain incompletely understood. Here, we performed all-atom molecular dynamics (MD) simulations of the human CFTR 3D structure in both the apo and VX-770 (ivacaftor)-bound states, embedded in a heterogeneous lipid bilayer, in order to systematically analyze electrostatic interactions, linking amino acids to each other as well as to anions and membrane lipids. We identified 557 electrostatic interactions between charged and polar amino acid side chains, which we systematically mapped across the CFTR 3D structure. They are organized into specific regions, with a subset showing high frequency and conservation across simulations, suggesting a structural role in stabilizing CFTR architecture. In contrast, more transient electrostatic interactions were detected in dynamic regions potentially linked to conformational transitions or other functional roles. Irregularities in transmembrane (TM) helices often incorporate amino acids involved in electrostatic interactions. Many basic and polar residues involved in electrostatic interactions also engaged in anion coordination, underscoring their contribution to ion conduction. In addition, some showed selective interactions with cholesterol and phosphatidylserine, revealing spatially organized lipid binding, particularly at the level of the lasso and in the vicinity of the VX-770 binding site, which may mark regions important for allosteric communication. VX-770 binding preserved the global architecture of the electrostatic interaction networks but induced subtle shifts, acting on specific salt bridges. Regardless of whether VX-770 is present or not, a secondary portal displayed between TM10/TM12 emerged from these MD simulations, in addition to the main TM4/TM6 portal, whose morphology and diameter are controlled by a fluctuating salt bridge. Two exit routes also appeared for the exit of anions toward the extracellular milieu. Altogether, our integrative analysis highlights how dynamic electrostatic networks, together with ion and lipid interactions, support CFTR's structural plasticity and functional modulation, offering molecular insights into potentiation mechanisms and into the specific evolution of CFTR in the ABC transporter superfamily.

Allostery plays a critical role in protein dynamics and is essential for many biological functions. Over the past decade, various computational approaches have been proposed for predicting allosteric sites. However, the strengths and weaknesses of each method are not well understood. In this study, we created two independent datasets that had not been used in selected computational protocols: a CAPASP-General subset comprising holo state allosteric proteins and a CAPASP-Unbound subset comprising apo state allosteric proteins. We then systematically evaluated the accuracy of five allosteric site prediction tools across five dimensions: sensitivity, specificity, F1-score, MCC value and ranking capability. The results indicated that the machine learning models PASSer and APOP, which are based on protein physicochemical properties, not only achieved the highest success rate in sensitivity prediction but also lead in average F1-score and MCC value. However, these models performed better with the CAPASP-General subset than with the CAPASP-Unbound subset, suggesting that the prediction models require further improvement. These findings could facilitate the selection of appropriate prediction models for different allosteric proteins and enhance our understanding of protein function and regulatory mechanisms.

Aberrant protein oligomerization is a hallmark of neurodegenerative disorders, yet the conformational and kinetic underpinnings of early aggregation remain poorly understood due to the inability of structural techniques to capture transient, low-abundance oligomeric intermediates. This necessitates the development of a methodology that can characterize the conformational states related to protein unfolding and thus allow for the investigation of the molecular mechanism responsible for disease progression. Here, we demonstrate how temperature-controlled nanoelectrospray ionization (TC-nESI) combined with high-resolution ion mobility-mass spectrometry (IM-MS), surface-induced dissociation (SID), and limited proteolysis can be used to define the misfolding and oligomerization landscape of bovine Cu/Zn superoxide dismutase (SOD1). This integrative approach enables real-time detection of coexisting intermediates, and captures molecular events including metal-induced stability, monomer unfolding and assembly into heterogeneous soluble oligomers. Our results reveal that both holo- and apo-SOD1 undergo dimer dissociation followed by monomer misfolding and assembly into heterogeneous non-native oligomers, and that slow thermal ramping promotes the accumulation of misfolded monomers and higher-order complexes. Apo-SOD1 that lacks stabilizing metal cofactors, forms more compact and kinetically distinct oligomers via monomeric, dimeric and trimeric intermediates. Proteolysis and heat-induced fragmentation identify loops V, VI, VII, and the C-terminus as key labile regions contributing to oligomer interface formation, predominantly through hydrophobic interactions. Our findings establish a mechanistically rich model for early aggregation and demonstrate the capability of TC-nESI-IM-MS to temporally and structurally resolve misfolding transitions and oligomeric populations in a single experiment. This platform provides a framework to dissect oligomerization pathways relevant to neurodegenerative diseases.

Acyl carrier proteins (ACPs) are dynamic, structurally conserved α-helical proteins central to many primary and secondary metabolic processes. Whilst prior engineering efforts have focused on strategic mutagenesis and "helix swaps", much of the ACP sequence design space remains underexplored. Here, we create diverse variants of the archetypal ACP subclass - AcpP - using a bespoke sequence-generating algorithm (ALGO-CP), which utilises a combined evolutionary and physicochemical design approach. Using ALGO-CP, we generated two soluble candidates - ALGO-055 and ALGO-059 - that can undergo full post-translational modification from apo → holo → acyl forms in vitro, using recombinant modifying enzymes. Building on these successful designs, we further adapted ALGO-CP to produce several chimeras, two of which - [ch]ALGO-012 and [ch]ALGO-024 - also exhibit full modifiability. We explore the structural plasticity of our ALGO variants via robust molecular dynamic simulations, and we further reveal by circular dichroism spectroscopy that ALGO-055 and ALGO-059 lack the canonical α-helical fold of an ACP, whilst remaining soluble and readily modifiable. Upon acylation of ALGO-055 and ALGO-059, we observe a marked increase in helicity indicative of protein restructuring. Of note, both ALGO-055 and ALGO-059 harbour several rare amino acid variations across their sequences, whilst preserving many important acidic "hotspots" involved in key protein-protein interactions. By testing the limits of the AcpP design space, our findings suggest that some key aspects of ACP behaviour (specifically post-translational modification) can be retained independently of the canonical structure. This work establishes a foundation for probing ACP sequence diversity through a hybrid computational-experimental approach. ALGO-CP is available under AGPL3.0 license: https://github.com/MAHerrera-94/ALGO_CP.

Despite adequate primary and secondary prevention of cardiovascular events, significant residual risk remains. Part of this risk has been attributed to lipoprotein(a) (Lp{a}). This is a genetically determined lipoprotein that has been linked to cardiovascular disease. Its variability and pathogenicity are attributed to the unique apolipoprotein(a) (apo{a}) within the molecule. This protein has been related to proatherogenic, proinflammatory, prothrombotic, and procalcific mechanisms that favor cardiovascular disease (CVD). Although it is recognized as a causal factor in disease, there are currently no approved therapeutics targeting this lipoprotein. Current management focuses on aggressive control of traditional cardiovascular risk factors. Novel therapeutics targeting Lp(a), including small interfering ribonucleic acids (siRNAs) and antisense oligonucleotides (ASOs), showed promising results in phase 2 trials. Multiple therapeutics are currently undergoing phase 3 trials, promising to bring a solution to this unsolved issue.

Flavoproteins are involved in a wide array of biological processes. These proteins contain one or more flavins as their cofactor, bound either noncovalently or covalently, and catalyze a wide breadth of redox reactions. In commonly used expression strains, flavoproteins are typically produced as holo (flavin-bound) flavoproteins. The ability to produce their apo form (flavoprotein devoid of flavins) will facilitate dedicated structural and mechanistic studies, while it also allows the incorporation of new-to-nature flavin-like cofactors. To facilitate this, we constructed, using the CRISPR/Cas9 system, an Escherichia coli strain that is impaired in producing the canonical FMN and FAD cofactors, due to a deletion in the ribB gene. This riboflavin auxotrophic strain is able to produce apoproteins of FMN- and FAD-dependent flavoproteins. We demonstrate that it can also be used for the incorporation of flavin derivatives by supplementing the medium with the respective riboflavin derivative. In parallel, we constructed, by genomic integration, a strain expressing an FAD synthetase from a T7 promoter and a flavin transferase from a lac promoter. This strain facilitates employing the newly developed methodology of flavin-tagging and flavin-fixing of target proteins, resulting in proteins carrying a covalently tethered FMN. It eliminates the need for two or more plasmids to generate covalently flavinylated flavoproteins. A third strain was prepared in which the features of riboflavin auxotrophy and flavin transferase activity were combined. This strain is perfectly suited for generating flavoproteins carrying a covalently anchored flavin derivative. These newly engineered strains, derivatives of E. coli BL21-AI, represent powerful tools for producing, investigating, and applying flavoproteins.

Photoaffinity labeling (PAL) utilizes photoreactive molecules to derivatize proteins in a solvent-accessible dependent manner. The site and frequency of the products of this reaction can be used to garner insights into the conformation of the protein. In this work, we document and characterize a novel oxidation process instigated by UV irradiation of aromatic diazirines. Diazirine is an increasingly utilized reagent for probing protein conformations, and this product has yet to be documented. We initially assess the selectivity of the chemical reaction and find that it is highly selective to methionine (Met) and tryptophan (Trp) residues. We next examine whether this oxidative labeling can be utilized to evaluate protein conformation. We assess native and urea-denatured ubiquitin and cytochrome c, holo- and apo-myoglobin, and native and copper-bound β-2-microglobulin. In all surveyed protein systems but ubiquitin, this diazirine-promoted oxidation readily differentiates between the two protein conformations.

Human methionine synthase (MTR) is an essential enzyme of one carbon metabolism. Consisting of a catalytic N-half and a cobalamin binding C-half, MTR utilises this intricate organometallic cofactor in the methyl transfer from methyltetrahydrofolate to homocysteine producing methionine. Cobalamin loading into MTR, and its subsequent activation, requires methylmalonic aciduria and homocystinuria Type D (MMADHC) protein and methionine synthase reductase (MTRR), respectively. However, the molecular basis of cobalamin binding and activation of human MTR aided by MMADHC and MTRR remains unknown. Here, using cryo-electron microscopy, we determine structures of human MTR in its apo, and cobalamin bound states. Apo MTR adopts a conformation where the two halves of the enzyme act independently with the C-half posed to bind cobalamin. Binding of cobalamin and its activation causes conformational changes in MTR that result in a flexible catalytically active state. AlphaFold predictions, validated by interaction studies, show that MMADHC interacts with the C-half of apo MTR to facilitate cobalamin loading. Unexpectedly we found that MTRR interacts at two distinct sites within the C-half of MTR which may aid in activation. Collectively these findings lay the groundwork to uncover the mechanisms through how MMADHC and MTRR coordinate cobalamin loading and activation of human MTR.