Identifying the molecular basis of Laminin N-terminal domain Ca2+ binding using a hybrid approach.

Ca2+ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca2+ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca2+ binding domain and in many cases requires Ca2+ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca2+ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca2+ interaction and identify a conserved sequence present in Ca2+ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca2+ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca2+ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca2+, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca2+ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca2+ dynamics within the context of the LN domain.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation.

NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer-dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.

Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor.

The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. KEY POINTS: • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality.

Heparin Mimetics and Their Impact on Extracellular Matrix Protein Assemblies.

Heparan sulfate is a crucial extracellular matrix component that organizes structural features and functional protein processes. This occurs through the formation of protein-heparan sulfate assemblies around cell surfaces, which allow for the deliberate local and temporal control of cellular signaling. As such, heparin-mimicking drugs can directly affect these processes by competing with naturally occurring heparan sulfate and heparin chains that then disturb protein assemblies and decrease regulatory capacities. The high number of heparan-sulfate-binding proteins that are present in the extracellular matrix can cause obscure pathological effects that should be considered and examined in more detail, especially when developing novel mimetics for clinical use. The objective of this article is to investigate recent studies that present heparan-sulfate-mediated protein assemblies and the impact of heparin mimetics on the assembly and function of these protein complexes.

The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation.

Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.

A Structural Analysis of Proteinaceous Nanotube Cavities and Their Applications in Nanotechnology.

Protein nanotubes offer unique properties to the materials science field that allow them to fulfill various functions in drug delivery, biosensors and energy storage. Protein nanotubes are chemically diverse, modular, biodegradable and nontoxic. Furthermore, although the initial design or repurposing of such nanotubes is highly complex, the field has matured to understand underlying chemical and physical properties to a point where applications are successfully being developed. An important feature of a nanotube is its ability to bind ligands via its internal cavities. As ligands of interest vary in size, shape and chemical properties, cavities have to be able to accommodate very specific features. As such, understanding cavities on a structural level is essential for their effective application. The objective of this review is to present the chemical and physical diversity of protein nanotube cavities and highlight their potential applications in materials science, specifically in biotechnology.

Boron rich nanotube drug carrier system is suited for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a two-step therapeutic process that utilizes Boron-10 in combination with low energy neutrons to effectively eliminate targeted cells. This therapy is primarily used for difficult to treat head and neck carcinomas; recent advances have expanded this method to cover a broader range of carcinomas. However, it still remains an unconventional therapy where one of the barriers for widespread adoption is the adequate delivery of Boron-10 to target cells. In an effort to address this issue, we examined a unique nanoparticle drug delivery system based on a highly stable and modular proteinaceous nanotube. Initially, we confirmed and structurally analyzed ortho-carborane binding into the cavities of the nanotube. The high ratio of Boron to proteinaceous mass and excellent thermal stability suggest the nanotube system as a suitable candidate for drug delivery into cancer cells. The full physicochemical characterization of the nanotube then allowed for further mechanistic molecular dynamic studies of the ortho-carborane uptake and calculations of corresponding energy profiles. Visualization of the binding event highlighted the protein dynamics and the importance of the interhelical channel formation to allow movement of the boron cluster into the nanotube. Additionally, cell assays showed that the nanotube can penetrate outer membranes of cancer cells followed by localization around the cells' nuclei. This work uses an integrative approach combining experimental data from structural, molecular dynamics simulations and biological experiments to thoroughly present an alternative drug delivery device for BNCT which offers additional benefits over current delivery methods.

Proteinaceous Nano container Encapsulate Polycyclic Aromatic Hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are toxic, mutagenic and among the most damaging chemical compounds with regard to living organisms. Because of their persistence and wide distribution removal from the environment is an important challenge. Here we report a new Nano container matrix based on the deep sea archaea-derived RHCC-Nanotube (RHCC-NT), which rapidly and preferentially binds low molecular weight PAHs. Under controlled-laboratory conditions and using fluorescence spectroscopy in combination with X-ray crystallography and MD simulations, we quantified the real-time binding of low molecular weight PAHs (2-4 rings) to our substrate. Binding coefficients ranged from 5.4 ± 1.6 (fluorene) to 32 ± 7.0 µM (acenaphthylene) and a binding capacity of 85 pmoles PAH per mg RHCC-NT, or 2.12 µmoles in a standard 25 mg sampler. The uptake rate of pyrene was calculated to be 1.59 nmol/hr∙mol RHCC-NT (at 10  C). Our results clearly show that RHCC-NT is uniquely suited as a monitoring matrix for low molecular weight PAHs.