Unfolding and refolding of the constant fragment of the immunoglobulin light chain

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. protein evolution | antibody structure | protein folding | protein engineering

Section: Resultsmentioning

confidence: 99%

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

Feige

Gräwert

Marcinowski

et al. 2014

“…The C L domain folds via an obligatory intermediate on two parallel pathways to its native state, the slower one being limited by the isomerization of the Y34-P35 bond to the native cis conformation (30,32). This bond is predominantly trans in the unfolded state.…”

Section: Resultsmentioning

confidence: 99%

“…This bond is predominantly trans in the unfolded state. As a consequence, only Ϸ10% of the molecules are able to fold to the native state within a few seconds (30,32), and Ϸ90% of the molecules have to undergo the intrinsically slow isomerization reaction before complete folding to the native state (30,32). At 2°C this reaction takes several hours to complete [see supporting information (SI) Fig.…”

Section: Resultsmentioning

confidence: 99%

The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity

Feige

Groscurth

Marcinowski

et al. 2008

Folding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate exhibits the basic ␤-barrel topology, yet some strands are distorted. Surprisingly, two short strand-connecting helices conserved in constant antibody domains assume their completely native structure already in the intermediate, thus providing a scaffold for adjacent strands. By transplanting these helical elements into ␤ 2 -microglobulin, a highly homologous member of the same superfamily, we drastically reduced its amyloidogenicity. Thus, minor structural differences in an intermediate can shape the folding landscape decisively to favor either folding or misfolding.amyloids ͉ NMR ͉ protein folding ͉ antibodies ͉ molecular dynamics

“…In unstructured protein chains, the trans form is favored over cis, and therefore proteins with cis prolyl bonds in the native state must undergo trans 3 cis isomerization during their folding (9, 10). In this case, folding typically starts with a particular proline still in the incorrect (trans) state, but when a certain extent of folding is reached, this trans proline acts as a barrier and blocks further folding (11-13).The coupling between conformational folding and prolyl isomerization has been studied for several small single-domain proteins (14)(15)(16)(17)(18)(19). In the folding of the gene-3-protein (G3P) of phage fd, prolyl isomerization determines the rate of the final domain assembly step.…”

mentioning

confidence: 99%

“…The coupling between conformational folding and prolyl isomerization has been studied for several small single-domain proteins (14)(15)(16)(17)(18)(19). In the folding of the gene-3-protein (G3P) of phage fd, prolyl isomerization determines the rate of the final domain assembly step.…”

mentioning

confidence: 99%

A remote prolyl isomerization controls domain assembly via a hydrogen bonding network

Weininger

Jakob²,

Eckert³

et al. 2009

Prolyl cis/trans isomerizations determine the rates of protein folding reactions and can serve as molecular switches and timers. In the gene-3-protein of filamentous phage, Pro-213 trans 3 cis isomerization in a hinge region controls the assembly of the 2 domains N1 and N2 and, in reverse, the activation of the phage for infection. We elucidated the structural and energetic basis of this proline-limited domain assembly at the level of individual residues by real-time 2D NMR. A local cluster of inter-domain hydrogen bonds, remote from Pro-213, is stabilized up to 3,000-fold by trans 3 cis isomerization. This network of hydrogen bonds mediates domain assembly and is connected with Pro-213 by rigid backbone segments. Thus, proline cis/trans switching is propagated in a specific and directional fashion to change the protein structure and stability at a distant position.gene-3-protein ͉ molecular timer ͉ proline isomerization ͉ protein folding ͉ real-time NMR T he cis/trans isomerizations of peptidyl-prolyl bonds in proteins are intrinsically slow processes. They determine the rates of protein folding reactions and are used as slow switches to regulate biological processes (1-8). In unstructured protein chains, the trans form is favored over cis, and therefore proteins with cis prolyl bonds in the native state must undergo trans 3 cis isomerization during their folding (9, 10). In this case, folding typically starts with a particular proline still in the incorrect (trans) state, but when a certain extent of folding is reached, this trans proline acts as a barrier and blocks further folding (11-13).The coupling between conformational folding and prolyl isomerization has been studied for several small single-domain proteins (14)(15)(16)(17)(18)(19). In the folding of the gene-3-protein (G3P) of phage fd, prolyl isomerization determines the rate of the final domain assembly step. G3P consists of 3 domains. The carboxy terminal (CT) domain anchors G3P in the phage coat (20), and the 2 amino terminal domains N1 and N2 form a functional entity that protrudes from the phage surface and mediates the infection of Escherichia coli cells (21). In the N1-N2 unit (Fig. 1A), N1 (residues 1-67) and N2 (residues 105-204) are linked by the hinge region, which forms numerous contacts with the N1 domain. The hinge region is formed by 2 noncontiguous chain regions (residues 89-104 between N1 and N2 and residues 205-220 after N2) and shows a well-ordered structure in the native protein.The time course of folding of G3P extends from milliseconds to hours (Fig. 1B). It starts with an extremely rapid folding reaction in the N1 domain with a time constant ϭ 9.4 ms, followed by 2 folding reactions in N2, which show values of 7 and 42 s. In the final, very slow step ( ϭ 6,200 s), the 2 domains assemble in a reaction that involves further folding in the hinge region and is limited in rate by the trans 3 cis isomerization of 22).When G3P is fully folded, the phage is not infectious, because the binding site for its receptor is buried at the int...