Mechanistic insights into the C-type lectin receptor CLEC12A-mediated immune recognition of monosodium urate crystal.

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.

Probing nanomechanical interactions of SARS-CoV-2 variants Omicron and XBB with common surfaces.

The emergence of SARS-CoV-2 variants has further raised concerns about viral transmission. A fundamental understanding of the intermolecular interactions between the coronavirus and different surfaces is needed to address the transmission of SARS-CoV-2 through respiratory droplet-contaminated surfaces or fomites. The receptor-binding domain (RBD) of the spike protein is a key target for the adhesion of SARS-CoV-2 on the surface. To understand the effect of mutations on adhesion, atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) was used to quantify the interactions between wild-type, Omicron, and XBB with several surfaces. The measurement revealed that RBD exhibits relatively higher forces on paper and gold surfaces, with the average force being 1.5 times greater compared to that on plastic surface. In addition, the force elevation on paper and gold surfaces for the variants can reach ∼28% relative to the wild type. These findings enhance our understanding of the nanomechanical interactions of the virus on common surfaces.

S373P Mutation Stabilizes the Receptor-Binding Domain of the Spike Protein in Omicron and Promotes Binding.

A cluster of several newly occurring mutations on Omicron is found at the ß-core region of the spike protein's receptor-binding domain (RBD), where mutation rarely happened before. Notably, the binding of SARS-CoV-2 to human receptor ACE2 via RBD happens in a dynamic airway environment, where mechanical force caused by coughing or sneezing occurs. Thus, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of RBDs and found that the mechanical stability of Omicron RBD increased by ∼20% compared with the wild type. Molecular dynamics (MD) simulations revealed that Omicron RBD showed more hydrogen bonds in the ß-core region due to the closing of the α-helical motif caused primarily by the S373P mutation. In addition to a higher unfolding force, we showed a higher dissociation force between Omicron RBD and ACE2. This work reveals the mechanically stabilizing effect of the conserved mutation S373P for Omicron and the possible evolution trend of the ß-core region of RBD.

Structure of Collagen.

Collagen is the most abundant fibrous protein in nature and widely exists in tissues such as connective tissue, tendon, skin, bone, and cartilage. On the one hand, collagen provides mechanical support in tissues, and on the other hand, plays an important role in controlling cell adhesion, cell migration, and tissue repair. A systematic understanding of the structure of collagen can promote the understanding of the biological functions of collagen scaffolds, and also provide theoretical guidance for applications of these natural fibrous protein materials. Therefore, this chapter centers on introducing the structure of collagen. As collagen has a typical hierarchical structure, the introduction to its structure will also be divided into different structural levels, from primary structure to quaternary structure. Due to the diversity of collagen types, this chapter will mainly focus on type I collagen.

Structure of Elastin.

As the extracellular matrix protein, elastin is a crucial component of connective tissue in life. It is responsible for the structural integrity and function of tissues undergoing reversible extensibility or deformability, even though it may make up only a small percentage of a tissue. The structure stability, elastic resilience, bioactivity, and ability of self-assembly make elastin a highly desirable candidate for the fabrication of biomaterials. Elastin's properties mainly depend on their special structure. As elastin can be obtained by the assembly and cross-linking of its soluble precursor, tropoelastin. This chapter centers on introducing the structure of those two materials.

Structure of Resilin.

Resilin, an insect structural protein, exhibits rubberlike elasticity characterized by low stiffness, high extensibility, efficient energy storage, exceptional resilience, and fatigue lifetime. The outstanding mechanical properties of native resilin have motivated recent research about resilin-like biomaterials for a wide range of applications. The systematic understanding of the resilin structure provides theoretical guidance for its applications. In this chapter, we systematically introduce its special structure, providing useful information for the structure and elastic mechanism of native resilin protein.

Recombination and Purification of Elastin-Like Polypeptides.

Elastin, as an extracellular matrix protein, has inherent advantages for biomedical applications. For example, it is highly extensible and biocompatible, biodegradable, and has no immunogenicity. However, directly extracting elastin from biological tissues remains challenging because they usually coexist with other proteins such as collagen. Therefore, an effective strategy to produce elastin is to transfer the elastin's target gene into other expression hosts and synthesize the resultant polypeptides using chemical biology methods. The polypeptides and proteins produced using these methods are usually referred to as elastin-like peptides (ELPs), which have received intensive interests in drug delivery and release, tissue engineering, implanted devices, and so on. Therefore, this chapter introduces the detailed protocol for the preparation of ELPs using genetic recombination, including DNA recombination, expression, and purification. The methods presented here are expected to provide methodological guidance for preparation and application of ELP materials.

Synthesis and Assembly of Recombinant Collagen.

Collagen represents the major structural protein of the extracellular matrix. The desired mechanical and biological performances of collagen that have led to its broad applications as a building block in a great deal of fields, such as tissue engineering, drug delivery, and nanodevices. The most direct way to obtain collagen is to separate and extract it from biological tissues, but these top-down methods are usually cumbersome, and the structure of collagen is usually destroyed during the preparation process. Moreover, there is currently no effective method to separate some scarce collagens (such as collagen from human beings). Alternatively, bottom-up assembly methods have been developed to obtain collagen assembly or their analogs. The collagen used in this type of method is usually obtained by genetic recombination. A distinct advantage of gene recombination is that the sequence structure of collagen can be directly customized, so its assembly mode can be regulated at the primary structure level, and then a collagen assembly with a predesigned configuration can be achieved. Additionally, insights into the assembly behavior of these specific structures provide a rational approach to understand the pathogenic mechanisms of collagen-associated diseases, such as diabetes. In this chapter, Type I collagen is used as an example to introduce the key methods and procedures of collagen recombination, and on this basis, we will introduce in detail the experimental protocols for further assembly of these recombinant proteins to specific structures, such as fibril.

Formation, Structure, and Mechanical Performance of Silk Nanofibrils Produced by Heat-Induced Self-Assembly.

The heat-induced self-assembly of silk fibroin (SF) is studied by combing fluorescence assessment, infrared nanospectroscopy, wide-angle X-ray scattering, and Derjaguin-Muller-Toporov coupled with atomic force microscopy. Several fundamental issues regarding the formation, structure, and mechanical performance of silk nanofibrils (SNFs) under heat-induced self-assembly are discussed. Accordingly, SF in aqueous solution is rod-like in shape and not micellar. The formation of SNFs occurs through nucleation-dependent aggregation, but the assembly period is variable and irregular. SF shows inherent fractal growth, and this trend is critical for the short-term assembly. The long-term assembly of SF, however, mainly involves an elongation growth process. SNFs produced by different methods, such as ethanol treatment and heat incubation, have similar secondary structure and mechanical properties. These investigations improve the in-depth understanding of fundamental issues related to self-assembly of SNFs, and thus provide inspiration and guidance in designing of silk nanomaterials.