乙型肝炎病毒前S2基因酵母表达载体的构建及表达

目的：探讨乙型肝炎病毒（HBV）前S2蛋白的功能。方法：以质粒pCP10 (含有HBV ayw亚型全长序列)为模板，多聚酶链反应（PCR）扩增HBV前S2基因，克隆到pGEM-T载休整 ，测序鉴定、酶切后回收，与酵母表达质粒pGBKT7连接。将重组载体转化酵母细胞AH109，提取酵母蛋白质，进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹分析。结果：成功构建HBV前S2基因酵母表达载体，Western免疫印迹显示HBV前S2蛋白在酵母细胞中表达，表达产物在胞内存在，分子量24kD左右。结论：HBV前S2蛋白在酵母细胞中表达成功。     

HBsAg诱饵载体的构建、表达及人胰腺cDNA文库的扩增、纯化和转化

目的构建HBsAg诱饵真核表达载体,并在AH109酵母菌中进行表达,同时扩增、纯化、鉴定及转化人胰腺cDNA文库。方法通过PCR扩增HBsAg基因,克隆到pGEM-T载体,测序正确后酶切并连接至表达载体pGBKT7中,转化酵母菌AH109,在色氨酸缺陷型培养基(SD/-Trp/Kana)上筛选阳性菌落,提取重组蛋白,经SDS-PAGE电泳后进行Western blot分析。扩增、纯化人胰腺cDNA文库并进行酶切鉴定及生物信息学分析,醋酸锂法将其转入Y187酵母菌。结果成功构建酵母表达载体pGBKT7-HBsAg、SDS-PAGE和Western blot显示重组蛋白在酵母细胞中正确表达;成功构建人胰腺cDNA文库,其容量为4×109 CFU/L,插入片段大小为0.5～2.0kb,长度不均,重组率为100%。结论成功构建HBsAg真核载体并在AH109酵母菌中表达,同时获得高质量的胰腺cDNA文库并转化入Y187酵母菌,为通过酵母双杂交筛选与HBsAg相互作用蛋白基因奠定了基础。     

乙型肝炎病毒核心抗原诱饵载体的构建及酵母表达

目的 构建乙型肝炎病毒核心抗原真核表达载体,并在酵母细胞中进行表达. 方法 以实验室保存质粒为模板,通过聚合酶链式反应(PCR)扩增HBcAg基因,克隆到pGEM-T载体.测序正确后酶切并连接至酵母表达载体pGBKT7中,转化酵母菌AH109,在色氨酸缺陷型培养基(SD/-Trp/Kana)上筛选阳性菌落后大量表达并提取重组蛋白,再进行SDS-PAGE和Western blot分析.结果 成功构建酵母表达载体pGBKT7-HBcAg,Western blot结果显示重组蛋白在酵母细胞中正确表达.结论 利用真核表达载体在酵母细胞中成功表达HBcAg蛋白,为研究HBV合并代谢性疾病的机制奠定了基础.     

乙型肝炎病毒e抗原真核表达载体的构建及在酵母中的表达

为探讨乙型肝炎病毒(HBV)e抗原(HBeAg)的功能，用多聚酶链反应(PCR)的方法以HBV ayw亚型全序质粒pCP10为模板扩增HBeAg基因，克隆到pGEM—T载体中扩增并测序，鉴定符合GenBank报告序列。用EcoRI和Pstl双酶切后回收片段，连接到真核表达载体pGBK17中并转化酵母AH109，在色氨酸缺陷型培养基(SD／—Trp)上筛选阳性菌落。提取阳性酵母菌的蛋白质，进行十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE)和Western免疫印迹分析，显示HBeAg基因在酵母细胞中表达，表达产物在胞内存在，相对分子质量(MT)为43000左右。     

禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。     

酵母双杂交诱饵蛋白LASS1融合表达质粒的构建及鉴定

目的 构建酵母双杂交系统诱饵蛋白大鼠LASS1融合表达质粒,为进一步筛选大鼠脑神经元内与LASS1p相互作用的蛋白奠定基础.方法 应用PCR方法获得大鼠LASS1基因2个片段,分别克隆入酵母双杂交系统诱饵蛋白质粒载体pGBKT7,转染酵母菌AH109并检测重组质粒的自激活现象及毒性.利用Western印迹检测重组质粒在AH109的表达情况.结果 LASS1基因的PCR产物片段大小分别为Flag(111 bp)、Slag(109 bp);Western印迹结果表明2个重组质粒表达的蛋白均可以与抗c-Myc抗体在22 kU处特异性反应;重组质粒转化酵母后无自激活作用.结论 重组质粒pGBKT7-Flag、pGBKT7-Slag均能够在AH109内正确表达,可作为酵母双杂交系统诱饵蛋白使用.     

丙型肝炎病毒F蛋白反式激活蛋白2基因在酵母细胞中的表达

目的为探讨丙型肝炎病毒(HCV)F蛋白反式激活蛋白2(HCV FTP2)的功能,在真核生物酵母细胞中表达HCV FTP2基因.方法以HepG2细胞来源的mRNA作为模板,经过逆转录聚合酶链反应(RT-PCR)扩增HCV FFP2基因,克隆到pGEM-T载体中,双酶切后回收连接到酵母表达质粒pGBKT7中表达.提取酵母蛋白质,进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot免疫印迹分析.结果成功构建HCV FTP2基因酵母表达载体,Western blot免疫印迹显示HCV FTP2基因在酵母细胞中表达成功.表达产物相对分子质量27kD.结论HCV FTP2在酵母中表达成功.     

丙型肝炎病毒NS3基因酵母表达载体构建及表达

目的　为探讨丙型肝炎病毒 (HCV)非结构蛋白NS3的功能 ,在真核生物酵母细胞中表达HCVNS3基因。方法　用聚合酶链反应 (PCR)的方法以HCV全长质粒pBRTM/HCV 1为模板扩增HCVNS3基因 ,克隆到 pGEM T载体中 ,双酶切后回收连接到酵母表达质粒 pGBKT7中表达。提取酵母蛋白质 ,进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析。结果　成功构建HCVNS3基因酵母表达载体 ,Western免疫印迹显示了HCVNS3在酵母细胞中表达。表达产物在胞内存在 ,相对分子质量为 70 0 0 0。结论　HCVNS3蛋白在酵母中表达成功。     

酵母双杂交技术筛选α干扰素结合蛋白的基因

目的 筛选人肝细胞cDNA文库中与α干扰素（IFNα）蛋白具有相互作用的蛋白基因。方法 用聚合酶链反应（PCR）扩增IFNα基因，连接入酵母表达载体pGBKT7中构建诱饵质粒，转染酵母细胞AH109，Western blot证明IFNα蛋白能够在AH109中表达。然后将AH109与转染了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合，在营养缺陷型培养基和X-α-半乳糖（X-α-gal）上进行双重筛选阳性菌落，提取质粒后转化DH5α大肠埃希菌并经氨苄西林抗性筛选，提取单克隆菌落质粒，酶切答定正确者进行测序，再行生物信息学分析。结果 成功克隆化IFNα基因并在酵母细胞中表达，应用酵母双杂交筛选出阳性菌落34个，经生物信息学分析，排除读码框架不正确者，最后得到8种已知基因：玻璃体连接蛋白、纤维蛋白原α多肽、人类免疫缺陷病毒（HIV）1Tat相互作用蛋白2、精氨酸酶、NADH脱氢酶1β亚复合物、转铁蛋白受体2α、酒精脱氢酶IBβ多肽、肝细胞癌蛋白（HCC-1）；2种染色体基因：人染色体17，克隆RP11-35083，人染色体10，克隆RP11-35101。结论 成功克隆出IFNα基因，并从肝细胞cDNA文库中筛选出8种能与IFNα具有结合作用的蛋白基因。     

酵母双杂交诱饵蛋白LASS1合表达质粒的构建及鉴定

目的构建酵母双杂交系统诱饵蛋白大鼠LASS1融合表达质粒，为进一步筛选大鼠脑神经元内与LASS1p相互作用的蛋白奠定基础。方法应用PCR方法获得大鼠LASS1基因2个片段，分别克隆人酵母双杂交系统诱饵蛋白质粒载体pGBKT7，转染酵母菌AH109并检测重组质粒的自激活现象及毒性。利用Western印迹检测重组质粒在AH109的表达情况。结果LASS1基因的PCR产物片段大小分别为Flag（111bp）、Slag（109bp）；Western印迹结果表明2个重组质粒表达的蛋白均可以与抗c-Myc抗体在22ku处特异性反应；重组质粒转化酵母后无自激活作用。结论重组质粒pGBKT7-Flag、pGBKT7-Slag均能够存AH109内正确表达，可作为酵母双杂交系统诱饵蛋白使用。     

A thermodynamic definition of protein domains

Protein domains are conspicuous structural units in globular proteins, and their identification has been a topic of intense biochemical interest dating back to the earliest crystal structures. Numerous disparate domain identification algorithms have been proposed, all involving some combination of visual intuition and/or structure-based decomposition. Instead, we present a rigorous, thermodynamically-based approach that redefines domains as cooperative chain segments. In greater detail, most small proteins fold with high cooperativity, meaning that the equilibrium population is dominated by completely folded and completely unfolded molecules, with a negligible subpopulation of partially folded intermediates. Here, we redefine structural domains in thermodynamic terms as cooperative folding units, based on m-values, which measure the cooperativity of a protein or its substructures. In our analysis, a domain is equated to a contiguous segment of the folded protein whose m-value is largely unaffected when that segment is excised from its parent structure. Defined in this way, a domain is a self-contained cooperative unit; i.e., its cooperativity depends primarily upon intrasegment interactions, not intersegment interactions. Implementing this concept computationally, the domains in a large representative set of proteins were identified; all exhibit consistency with experimental findings. Specifically, our domain divisions correspond to the experimentally determined equilibrium folding intermediates in a set of nine proteins. The approach was also proofed against a representative set of 71 additional proteins, again with confirmatory results. Our reframed interpretation of a protein domain transforms an indeterminate structural phenomenon into a quantifiable molecular property grounded in solution thermodynamics.     

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1’s involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ_lag) and maximum growth rate (ν_max) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ_lag/∂[urea] and ∂ log ν_max/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.     

Experimental support for the evolution of symmetric protein architecture from a simple peptide motif

The majority of protein architectures exhibit elements of structural symmetry, and "gene duplication and fusion" is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique "top-down symmetric deconstruction" strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed "conserved architecture" model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.     

Retro-translocation of mitochondrial intermembrane space proteins

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.Mitochondrial biogenesis is essential for eukaryotic cells. Because most mitochondrial proteins originate in the cytosol, mitochondria had to develop a protein import system. Given the complex architecture of these organelles, with two membranes and two aqueous compartments, protein import and sorting require the cooperation of several pathways. The main entry gate for precursor proteins is the translocase of the outer mitochondrial membrane (TOM) complex. Upon entering mitochondria, proteins are routed to different sorting machineries (1–5).Reaching the final location is one step in the maturation of mitochondrial proteins that must be accompanied by their proper folding. The mitochondrial intermembrane space assembly (MIA) pathway for intermembrane space (IMS) proteins illustrates the importance of coupling these processes because this pathway links protein import with oxidative folding (6–10). Upon protein synthesis in the cytosol, the cysteine residues of IMS proteins remain in a reduced state, owing to the reducing properties of the cytosolic environment (11, 12). After entering the TOM channel, precursor proteins are specifically recognized by Mia40 protein, and their cysteine residues are oxidized through the cooperative action of Mia40 and Erv1 proteins (7, 13–17). Mia40 is a receptor, folding catalyst, and disulfide carrier, and the Erv1 protein serves as a sulfhydryl oxidase. The oxidative folding is believed to provide a trapping mechanism that prevents the escape of proteins from the IMS back to the cytosol (10, 13, 18). Our initial result raised a possibility that the reverse process can also occur, as we observed the relocation of in vitro imported Tim8 from mitochondria to the incubation buffer (13). Thus, we sought to establish whether and how this process can proceed in the presence of the intact outer membrane (OM). Our study provides, to our knowledge, the first characterization of the mitochondrial protein retro-translocation. The protein retro-translocation serves as a regulatory and quality control mechanism for the mitochondrial IMS proteome.     

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

GTPases of immunity-associated proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of a representative member, GIMAP2, in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide-binding domain of the TRAFAC (translation factor associated) class with a unique amphipathic helix α7 packing against switch II. In the absence of α7 and the presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide-binding site, which also involves the GIMAP specificity motif and the nucleotide base. Structural rearrangements in switch II appear to induce the release of α7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. Although earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we reexamined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.     

Folding pathways of proteins with increasing degree of sequence identities but different structure and function

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called G_A (a 3-α helix fold) and G_B (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the G_A domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the G_Bs, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.     

Functional modulation of a protein folding landscape via side-chain distortion

Ultrahigh-resolution (< 1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6° distortion along the side-chain, estimated to cost 4 kcal/mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.     

Multimolecule test-tube simulations of protein unfolding and aggregation

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.     

Golden triangle for folding rates of globular proteins

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured rates of spontaneous folding of single-domain globular proteins range from microseconds to hours: the difference (11 orders of magnitude) is akin to the difference between the life span of a mosquito and the age of the universe. Here, we show that physical theory with biological constraints outlines a “golden triangle” limiting the possible range of folding rates for single-domain globular proteins of various size and stability, and that the experimentally measured folding rates fall within this narrow triangle built without any adjustable parameters, filling it almost completely. In addition, the golden triangle predicts the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control. It also predicts the maximal allowed size of the “foldable” protein domains, and the size of domains found in known protein structures is in a good agreement with this limit.     

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.The phylogenetically oldest living organisms identified that possess most major components of a vertebrate adaptive immune system are cartilaginous fish (Chondrichthyes) such as sharks, skates, and rays (1, 2). They shared the last common ancestor with other jawed vertebrates roughly 500 million years ago (2, 3). Accordingly, shark antibodies can provide unique insights into the molecular evolution of the immune system. Furthermore, shark antibodies have evolved under challenging conditions; for example, the high osmolarity of shark blood is partially sustained by the protein denaturant urea (4, 5). Even though it is partially counteracted by other osmolytes (6), shark antibodies are believed to be particularly stable (7). Insights into the structural features that provide this increased stability may provide attractive applications for biotechnology (8). Sharks and other Elasmobranchs have two conventional antibodies, IgM and IgW, but the structurally simplest antibody molecule in sharks is the so-called Ig new antigen receptor (IgNAR) (9). In its secreted form, it consists of two identical heavy chains (HCs) composed of one variable domain (V) and five constant domains (C1–C5) each (4, 9) (Fig. 1A). Similar to camelid antibodies, IgNARs are devoid of light chains (LCs) (9, 10), an example of convergent evolution (11). The variable domain of IgNAR, whose structure had been solved (12, 13), shows similarity to the variable domains of evolutionarily more recent immunoglobulins (12, 13). In contrast, its constant domains (C1–C5; Fig. 1A) are most homologous to the primordial IgW of sharks (14). Of the five human antibody classes, IgA, IgD, IgE, IgG, and IgM, IgW is most closely related to IgD, which, along with IgM, are the oldest Ig isotypes (14–17). Except for low-resolution electron microscopic images (18), no structural data are available for any of the constant IgNAR domains.Open in a separate windowFig. 1.Sequence and structure of IgNAR domains C1–C4. (A) Schematic of the secreted dimeric IgNAR molecule, comprising one variable (V) and five constant (C1–C5) domains. Predicted glycosylation sites are shown as gray hexagons. Cysteines that are not part of the intradomain disulfide bridges are indicated (–SH). The secretory tail is C terminally of the C5 domain. (B) Sequence alignment of IgNAR C1–C5 with the human IgG1 HC domains C_H1–C_H3. Conserved cysteines are highlighted in red, and conserved hydrophobic residues of a YxCxY (Y, hydrophobic residue) motif around the disulfide bridge are highlighted in orange. Conserved tryptophans in strand c and the second helix are highlighted in blue, and the cis-proline residue in the loop between strand b and c is depicted in cyan. Secondary structure elements are indicated above the alignment. Black arrows indicate strictly conserved residues, and gray arrows homologous residues. (C) Ribbon diagram of the isolated constant IgNAR domains C1–C4 (C1, cyan; C2, blue; C3, red; C4, green; colors like in A). Residues marked in the alignment are shown in stick representation, the small helices are indicated. (D) Superposition of the IgNAR C1-4 domains (C1, cyan; C2, blue; C3, red; C4, green) on a human IgG C_H3 domain (gray, Protein Data Bank ID code 1HZH).Here, we determined the structures of the four N-terminal IgNAR constant domains (C1–C4) at atomic resolution and present a model for the complete IgNAR molecule that reveals key adaptations of HC-only antibodies. We identified structural elements that contribute to the high stability of some IgNAR domains and transferred these to human antibodies to improve their stability and secretion.