However, the amino acid residues at the positions that determine the typical immunoglobulin fold are all well-conserved in the VHH [13,14]

However, the amino acid residues at the positions that determine the typical immunoglobulin fold are all well-conserved in the VHH [13,14]. The VHH domain Piroxicam (Feldene) consists of four framework regions (FR1, FR2, FR3, FR4), which are separated by three hypervariable loops, known as the complementarity-determining Rabbit Polyclonal to Ik3-2 region loops (CDRs), namely the CDR1, CDR2, and CDR3 loops (Figure 1). the simulations with and without NOE time-averaged restraints. We also compare the measured and calculated order parameters and find generally good agreement for the motions observed in the psCns timescale, in particular for the CDR3 loop. Understanding of the CDR3 loop dynamics is especially critical for nanobodies, as this loop is typically critical for antigen recognition. Keywords: single-domain Piroxicam (Feldene) antibody, nanobody, NMR, molecular dynamics simulations 1. Introduction Camelids such as camels, dromedaries, llamas, alpacas, guanacos, and vicu?as contain Piroxicam (Feldene) heavy-chain-only antibodies, which consist of a stable and soluble single-antigen-binding variable domain [1,2,3]. Single-domain antibodies (VHHs), also known as nanobodies?, have received increasing attention as highly versatile proteins with a high affinity for a variety of targets, and their flexibility has opened the door for a new generation of therapeutics [4,5]. Nanobodies have been proposed as treatments for various diseases and infections, including: autoimmune diseases, allergies, and for use as antivirals. The term nanobody originates from a trademark introduced by the company Ablynx in 2003 and became a general classification for these single-immunoglobulin domain proteins, reflecting their small size compared to antibodies, which are more than 10 times larger [6]. Nanobodies are potent alternatives to conventional antibodies, because of their small size, refolding capacity, stability, specificity, and natural origin [4,7,8]. Structurally, nanobodies are still functional without a light-chain counterpart found in typical antibodies, as they lack the hydrophobic interface, which is usually required to pair with a light chain in IgG-type antibodies [9]. Thus, the amino acid sequences of naturally occurring VHH antibodies are expected to contain adaptations to compensate for the absence of the paired light-chain variable domain. Nevertheless, it has been shown that VHH sequences share a high degree of similarity (~80%) with conventional human variable heavy-chain domains (VH). However, the less hydrophobic interface in VHH originates from several residue substitutions (mainly hydrophilic residues), namely L11S, V37F/Y, G44E, L45R/C, and W47G (following the Kabat nomenclature [10]), that discriminate the conventional VH from VHH. These residue substitutions Piroxicam (Feldene) are believed to enhance the stability in the absence of the light chain and result in favorable biophysical characteristics, such as stability and low aggregation risk [11,12]. However, the amino acid residues at the positions that determine the typical immunoglobulin fold are all well-conserved in the VHH [13,14]. The VHH domain consists of four framework regions (FR1, FR2, FR3, FR4), which are separated by three hypervariable loops, known as the complementarity-determining region loops (CDRs), namely the CDR1, CDR2, and CDR3 loops (Figure 1). The antigen-binding site, the paratope, is formed not exclusively by the CDR loops but also by neighboring framework residues, which contribute to recognizing and binding the antigen. VHHs contain a canonical disulfide bond connecting the -strands of framework regions 1 and 3. Various camelid antibodies also have an additional disulfide bond connecting either the end of the CDR1 loop with the CDR3 loop (camels) or the beginning of the CDR2 loop with the CDR3 loop (llamas). Furthermore, the CDR3 loop of VHHs can be substantially longer compared to conventional IgG and possesses the unique ability to form long extensions to reach cavities and buried binding sites with high shape complementarity [15]. Piroxicam (Feldene) Thus, the CDR3 loop of VHHs plays a critical role in recognizing and binding the antigen [16]. However, due to the high diversity in the length, sequence, and structure of the CDR3 loop, structure prediction remains challenging [17]. Recent studies using molecular dynamics (MD) simulations found that one single static structure is not sufficient to functionally understand the antigen-binding site, and suggested to characterize the paratopes as ensembles in solution [18,19,20]. We previously showed that antigen recognition follows a conformational selection-type binding to the dominant structure in solution, which is frequently not reflected by.