Ertan Eryilmaz

Naturally split inteins assemble through a "capture and collapse" mechanism

Split inteins are a class of naturally occurring proteins that carry out protein splicing in trans. The chemical mechanism of protein trans-splicing is well-understood and has been exploited to develop several powerful protein engineering technologies. Split intein chemistry is preceded by efficient molecular recognition between two protomers that become intertwined in their bound state. It is currently unclear how this unique topology is achieved upon fragment association. Using biophysical techniques in conjunction with protein engineering methods, including segmental isotopic labeling, we show that one split intein fragment is partly folded, while the other is completely disordered. These polypeptides capture each other through their disordered regions and form an ordered intermediate with native-like structure at their interface. This intermediate then collapses into the canonical intein fold. This mechanism provides insight into the evolutionary constraints on split intein assembly and should enhance the development of split intein-based technologies.

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

Background: The mechanism by which antibody constant region alters fine specificity is unknown. Results: Different constant regions were found to change electronic and chemical properties of the antigen-binding site. Conclusion: Constant regions can affect the energy landscape of the variable region. Significance: These results are potentially critical for understanding fast, correct immune responses at the systems level and for future immunotherapy development. The finding that the antibody (Ab) constant (C) region can influence fine specificity suggests that isotype switching contributes to the generation of Ab diversity and idiotype restriction. Despite the centrality of this observation for diverse immunological effects such as vaccine responses, isotype-restricted antibody responses, and the origin of primary and secondary responses, the molecular mechanism(s) responsible for this phenomenon are not understood. In this study, we have taken a novel approach to the problem by probing the paratope with 15N label peptide mimetics followed by NMR spectroscopy and fluorescence emission spectroscopy. Specifically, we have explored the hypothesis that the C region imposes conformational constraints on the variable (V) region to affect paratope structure in a V region identical IgG1, IgG2a, IgG2b, and IgG3 mAbs. The results reveal isotype-related differences in fluorescence emission spectroscopy and temperature-related differences in binding and cleavage of a peptide mimetic. We conclude that the C region can modify the V region structure to alter the Ab paratope, thus providing an explanation for how isotype can affect Ab specificity.

Extein Residues Play an Intimate Role in the Rate-Limiting Step of Protein Trans-Splicing

Split inteins play an important role in modern protein semisynthesis techniques. These naturally occurring protein splicing domains can be used for in vitro and in vivo protein modification, peptide and protein cyclization, segmental isotopic labeling, and the construction of biosensors. The most well-characterized family of split inteins, the cyanobacterial DnaE inteins, show particular promise, as many of these can splice proteins in less than 1 min. Despite this fact, the activity of these inteins is context-dependent: certain peptide sequences surrounding their ligation junction (called local N- and C-exteins) are strongly preferred, while other sequences cause a dramatic reduction in the splicing kinetics and yield. These sequence constraints limit the utility of inteins, and thus, a more detailed understanding of their participation in protein splicing is needed. Here we present a thorough kinetic analysis of the relationship between C-extein composition and split intein activity. The results of these experiments were used to guide structural and molecular dynamics studies, which revealed that the motions of catalytic residues are constrained by the second C-extein residue, likely forcing them into an active conformation that promotes rapid protein splicing. Together, our structural and functional studies also highlight a key region of the intein structure that can be re-engineered to increase intein promiscuity.

Structural and dynamical features of inteins and implications on protein splicing.

Protein splicing is a posttranslational modification where intervening proteins (inteins) cleave themselves from larger precursor proteins and ligate their flanking polypeptides (exteins) through a multistep chemical reaction. First thought to be an anomaly found in only a few organisms, protein splicing by inteins has since been observed in microorganisms from all domains of life. Despite this broad phylogenetic distribution, all inteins share common structural features such as a horseshoe-like pseudo two-fold symmetric fold, several canonical sequence motifs, and similar splicing mechanisms. Intriguingly, the splicing efficiencies and substrate specificity of different inteins vary considerably, reflecting subtle changes in the chemical mechanism of splicing, linked to their local structure and dynamics. As intein chemistry has widespread use in protein chemistry, understanding the structural and dynamical aspects of inteins is crucial for intein engineering and the improvement of intein-based technologies.

Global structures of IgG isotypes expressing identical variable regions.

Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG₃ Fab crystal structure from the same family was determined to 2.45 Å resolution. The IgG₃ Fab crystal structure differs from a closely related previously solved IgG1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG₃ was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure.

Nuclear spin relaxation in isotropic and anisotropic media.

2010 Elsevier B.V. All rights reserved.

The constant region affects antigen binding of antibodies to DNA by altering secondary structure

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

Structure and Dynamics of the P7 Protein from the Bacteriophage ϕ12

Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multiprotein polymerase complex (PX) to replicate and transcribe the viral genome. Although the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior is understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Toward obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus phi 12 at a resolution of 1.8 A. The N-terminal core region (1-129) of P7 forms a novel homodimeric alpha/beta-fold having structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results, combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly, hint toward its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130-169) that is essential for virus viability, although highly disordered, contains a nascent helix. We demonstrate for the first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminal tail of P7 play a role in RNA recognition. This interaction leads to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7-RNA interactions.

Anti-DNA antibody mediated catalysis is isotype dependent

Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies.

A peptide mimic blocks the cross-reaction of anti-DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.