Vladimir M. Tischenko

Resolving the energy paradox of chaperone/usher-mediated fibre assembly

Periplasmic chaperone/usher machineries are used for assembly of filamentous adhesion organelles of Gram-negative pathogens in a process that has been suggested to be driven by folding energy. Structures of mutant chaperone-subunit complexes revealed a final folding transition (condensation of the subunit hydrophobic core) on the release of organelle subunit from the chaperone-subunit pre-assembly complex and incorporation into the final fibre structure. However, in view of the large interface between chaperone and subunit in the pre-assembly complex and the reported stability of this complex, it is difficult to understand how final folding could release sufficient energy to drive assembly. In the present paper, we show the X-ray structure for a native chaperone-fibre complex that, together with thermodynamic data, shows that the final folding step is indeed an essential component of the assembly process. We show that completion of the hydrophobic core and incorporation into the fibre results in an exceptionally stable module, whereas the chaperone-subunit pre-assembly complex is greatly destabilized by the high-energy conformation of the bound subunit. This difference in stabilities creates a free energy potential that drives fibre formation.

Three-Dimensional Structure of the Human Myeloma IgG2

Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit) and CH2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of antibody-based therapeutics.

Long-term metastable conformation of human Fcγ subunit

Abstract It was found that the human (hu) myeloma IgG1 Ser, its Fcγ fragment and the chimeric mouse-human monoclonal antibody (chim-mAb), containing the constant part of hu-γ1-chain, can exist in a long-term metastable conformational state. This state arises as a result of short incubation of IgG molecules and their Fcγ fragments at pH γ 2 domains is unfolded, but rapidly refolds after neutralisation. At the same time, non-covalent interactions between C γ 2 and C γ 3 domains are restored very slowly. A metastable state of IgG keeps 70% of complement-binding ability in comparison with the native state.

Core hinge of human immunoglobulin G3 as a system of four independent co-operative blocks

On the heat absorption curves of human immunoglobulin G3 (hIgG3) Kuc melting the scanning calorimetry method reveals a high-temperature (high-T(m)) peak of high intensity that is absent at the curves of other hIgG subclasses and IgG of other species. An analogous peak is observed also at the curves of melting of hIgG3 fragments containing the hinge segments. The high-T(m) peak is accompanied by characteristic changes in circular dichroism (CD) spectra at 220-230 nm. This allows relating the peak to the melting of a poly-L-proline conformation of an extremely long hIgG3 core hinge. The comparison of deltaH(cal) and deltaH(eff) testifies that the core hinge can be considered as a system of four independent co-operative blocks connected by flexible sites. These sites may provide additional flexibility to the hIgG3 molecule and also permit a transition of the rod-like shape of the hinge to compact globule-like conformation.

Effect of hinge region state on interaction of human IgG3 with the complement system.

Covalently cross-linked rod-like dimers of human myeloma IgG3 are more efficient in the inhibition of the complement system reaction than compact dimers. This correlates with an increase in the stability of CH2 domains of the immunoglobulin in the state with the rod-like hinge region.

Destabilization of CH2 domains in intact IgG2 is accompanied by reduced ability to inhibit complement system factor C1.

Fc fragments (hFc) of human myeloma IgG2 proteins LOM and SIN having core hinge (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys) were first obtained by a modified proteolytic procedure. The thermostability of CH2 domains inside of standard Fc, hFc fragments, and intact IgG2 LOM and SIN was studied by fluorescence spectroscopy. It was found that CH2 domains of intact IgG2 are destabilized. The destabilization is accompanied by reduced ability of IgG2 to inhibit the activation of complement system by classical pathway. This could be due to the decrease in the affinity of CH2 domains to factor C1q.

Human myeloma IgG4 reveals relatively rigid asymmetric Y-like structure with different conformational stability of C H 2 domains

Graphical abstract Figure. No Caption available. HighlightsA 3D model of myeloma hIgG4 was created at ˜3 nm resolution using electron microscopy.The hIgG4 model reveals relatively rigid asymmetric Y‐like structure.One Fab subunit of hIgG4 is closer to the upper portion of the Fc subunit (CH2 domain) than the other Fab. ABSTRACT Human IgG4 (hIgG4) has weak pro‐inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ˜3 nm resolution using electron microscopy (EM) with negative staining and single‐particle 3D reconstruction. The hIgG4 model reveals relatively rigid asymmetric Y‐like structure. The model shows that one Fab subunit is closer to the upper portion of the Fc subunit (CH2 domain) than the other Fab. This is in agreement with X‐ray crystallography and X‐ray/neutron scattering, recently published by others. The same hIgG4 sample was studied with differential scanning calorimetry (DSC) and fluorescence. The thermodynamics and fluorescence observations indicate that one CH2 domain displays less conformational stability than the other. This finding is consistent with the flipping of one CH2 domain, observed in pembrolizumab (recombinant hIgG4) by X‐ray crystallography. The specific feature of hIgG4 CH2 domains together with relatively rigid asymmetric Y‐like structure, in which one Fab subunit is closer to the upper portion of the Fc subunit (CH2 domain) than the other Fab, can explain the unique biological properties of hIgG4, such as its weak pro‐inflammatory activity.

Human myeloma immunoglobulins of the fourth subclass (IgG4 MAM) contain a fraction with different properties of CH2 domains

A long-lived metastable minor fraction has been detected and characterized in myeloma protein IgG4 MAM by hydro- and thermodynamic methods. The sedimentation constants of the minor and the major protein fractions are different. The stability of the two CH2 domains in the minor fraction varies. The unique characteristics of these IgG4 MAM conformers arise from the fact that on exchange of the heavy chains between IgG4 molecules, in some of them only one non-canonical bond Cys226-Cys229 is formed in the central part of the “hinge region” instead of two canonical interchain disulfide bonds Cys226-Cys226 and Cys229-Cys229. This leads to asymmetric structure of the IgG4 MAM molecules.