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P., Delehanty J. germ collection V1 sequence, DPK9. The producing huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Allyl methyl sulfide Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-? resolution, respectively. The huE06 v1.1 molecule retained all but one amino Allyl methyl sulfide acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen conversation. E06 interacts with HSA in an atypical mode that utilizes considerable framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the functions of various elements of the molecule are explained with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen acknowledgement and provides a framework for further design and humanization of shark IgNARs. half-life of the molecules. They can also be linked to Fc domains of traditional antibodies to provide them with desired effector functions. IgNARs were discovered in sharks in the 1990s (7, 8). Their variable regions (V-NARs) are small (12C13-kDa), independently folding domains that demonstrate high biophysical stability, solubility, and ability to bind to a variety of antigens including epitopes located in clefts on protein surfaces (enzyme active sites) that are non-accessible by traditional antibody variable domains (9, 10). A similar preference for cleft acknowledgement was exhibited for camelid VHH antibodies (11C14). In both cases, the key to such acknowledgement is the structural business of the CDR loops, in particular CDR3, which is usually often long (15C18 residues) and Allyl methyl sulfide protruding from your V-NAR or VHH surface. V-NARs are unique from common Ig VH and VL domains as well as camelid VHH domains, sharing higher structural homology to immunoglobulin VL and T-cell receptor V domains than with immunoglobulin VH. The most unique feature of V-NARs is the absence of a CDR2 loop and of two -strands, C and C, associated with it. Instead, a distinct belt is created around the middle of the -sandwich structure (10, 15). This region shows an elevated rate of somatic mutations and has thus been termed hypervariable region 2 (HV2) (16). Another region of increased mutation frequency is located between HV2 and CDR3, comprising a loop that links -strands D and E comparable to that in T-cell receptor V chains; thus, this region was termed HV4. Structurally, HV2 is usually most proximal to CDR3, whereas HV4 is in proximity to CDR1. Several structural types of IgNAR variable domains have been classified based on the number and position of extra cysteine residues in CDRs and frameworks (FWs) in addition to the canonical cysteine pair (Cys23/Cys88 for VL; Kabat nomenclature) of the Ig fold (5). Type I V-NAR, found in nurse sharks, has 2 cysteines in CDR3 and 2 more cysteines in frameworks (FW2 and FW4). The more common type II has one extra cysteine pair, which links CDR1 and CDR3. Type III, detected primarily in neonatal shark development, is similar to type II but has a conserved Allyl methyl sulfide Trp residue in CDR1 and limited CDR3 diversity. Another structural type of V-NAR, which we have termed type IV, has only two canonical cysteine residues. So far, this type has been found primarily in dogfish sharks (Ref. 17 and this study) and was also isolated from semisynthetic V-NAR libraries derived from wobbegong sharks (18). The single domain nature and the lack of CDR2 in V-NARs heighten the requirement for CDR1 and CDR3 to provide specific and high affinity binding to prospective antigens. CDR3, which is usually more variable in terms of sequence, length, and conformation, plays the key role in antigen acknowledgement. The placing of cysteine residues in different V-NAR types is usually important for determining the conformation of CDRs. For example, CDR3 is long Rabbit polyclonal to ECHDC1 Allyl methyl sulfide and extended (and tethered to CDR1) in PBLA8, a type II V-NAR, which enables it to access the active site cavity of its target, hen egg white lysozyme (HEL; Ref. 10). In contrast, 5A7, a type I V-NAR also directed against lysozyme and targeting a similar surface epitope, has a long CDR3 that adopts a bent conformation and forms a rather flat binding surface that does not enter deep into the HEL active site (10, 15). Nevertheless, both HEL binders form comparable buried surface area with their target (700 ?2) and bind with low nanomolar affinity. The extent of the surface area is similar to values observed for the complexes of heavy chains of classical antibodies with their targets (19, 20). Besides CDR1 and CDR3, a few other.

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