Functional and structural insights into the multi-step activation and catalytic mechanism of bacterial ExoY nucleotidyl cyclase toxins bound to actin-profilin.

ExoY virulence factors are members of a family of bacterial nucleotidyl cyclases (NCs) that are activated by specific eukaryotic cofactors and overproduce cyclic purine and pyrimidine nucleotides in host cells. ExoYs act as actin-activated NC toxins. Here, we explore the Vibrio nigripulchritudo Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) ExoY effector domain (Vn-ExoY) as a model for ExoY-type members that interact with monomeric (G-actin) instead of filamentous (F-actin) actin. Vn-ExoY exhibits moderate binding affinity to free or profilin-bound G-actin but can capture the G-actin:profilin complex, preventing its spontaneous or VASP- or formin-mediated assembly at F-actin barbed ends in vitro. This mechanism may prolong the activated cofactor-bound state of Vn-ExoY at sites of active actin cytoskeleton remodelling. We present a series of high-resolution crystal structures of nucleotide-free, 3'-deoxy-ATP- or 3'-deoxy-CTP-bound Vn-ExoY, activated by free or profilin-bound G-actin-ATP/-ADP, revealing that the cofactor only partially stabilises the nucleotide-binding pocket (NBP) of NC toxins. Substrate binding induces a large, previously-unidentified, closure of their NBP, confining catalytically important residues and metal cofactors around the substrate, and facilitating the recruitment of two metal ions to tightly coordinate the triphosphate moiety of purine or pyrimidine nucleotide substrates. We validate critical residues for both the purinyl and pyrimidinyl cyclase activity of NC toxins in Vn-ExoY and its distantly-related ExoY from Pseudomonas aeruginosa, which specifically interacts with F-actin. The data conclusively demonstrate that NC toxins employ a similar two-metal-ion mechanism for catalysing the cyclisation of nucleotides of different sizes. These structural insights into the dynamics of the actin-binding interface of actin-activated ExoYs and the multi-step activation of all NC toxins offer new perspectives for the specific inhibition of class II bacterial NC enzymes.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

Iron-sulfur biology invades tRNA modification: the case of U34 sulfuration.

Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

Insight into microtubule nucleation from tubulin-capping proteins.

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or ß-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

Macromolecular interactions in vitro, comparing classical and novel approaches.

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.

Molecular basis for transfer RNA recognition by the double-stranded RNA-binding domain of human dihydrouridine synthase 2.

Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouridine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.

Characterization of the binding mode of JNK-interacting protein 1 (JIP1) to kinesin-light chain 1 (KLC1).

JIP1 was first identified as scaffold protein for the MAP kinase JNK and is a cargo protein for the kinesin1 molecular motor. JIP1 plays significant and broad roles in neurons, mainly as a regulator of kinesin1-dependent transport, and is associated with human pathologies such as cancer and Alzheimer disease. JIP1 is specifically recruited by the kinesin-light chain 1 (KLC1) of kinesin1, but the details of this interaction are not yet fully elucidated. Here, using calorimetry, we extensively biochemically characterized the interaction between KLC1 and JIP1. Using various truncated fragments of the tetratricopeptide repeat (TPR) domain of KLC1, we narrowed down its JIP1-binding region and identified seven KLC1 residues critical for JIP1 binding. These isothermal titration calorimetry (ITC)-based binding data enabled us to footprint the JIP1-binding site on KLC1-TPR. This footprint was used to uncover the structural basis for the marginal inhibition of JIP1 binding by the autoinhibitory LFP-acidic motif of KLC1, as well as for the competition between JIP1 and another cargo protein of kinesin1, the W-acidic motif-containing alcadein-α. Also, we examined the role of each of these critical residues of KLC1 for JIP1 binding in light of the previously reported crystal structure of the KLC1-TPR:JIP1 complex. Finally, sequence search in eukaryotic genomes identified several proteins, among which is SH2D6, that exhibit a motif similar to the KLC1-binding motif of JIP1. Overall, our extensive biochemical characterization of the KLC:JIP1 interaction, as well as identification of potential KLC1-binding partners, improves the understanding of how this growing family of cargos is recruited to kinesin1 by KLC1.

Dissociation of the Dimer of the Intrinsically Disordered Domain of RNase Y upon Antibody Binding.

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b λx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of α-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form.

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.

αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design.

αRep refers to a new family of artificial proteins based on a thermostable α-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to accommodate metal complexes and thus appears to be suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen, and independently changed into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine residue of each protein variant, and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was specifically bound by the two biohybrids with two different binding modes. Furthermore, the holo-biohybrid A3F119NPH was found to be capable of enantioselectively catalyzing Diels-Alder (D-A) cycloadditions with up to 62 % ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a promising new route for the design and production of new enantioselective biohybrids based on entirely artificial proteins obtained from a highly diverse library.

Power of protein/tRNA functional assembly against aberrant aggregation.

Understanding the mechanisms of protein oligomerization and aggregation is a major concern for biotechnology and medical purposes. However, significant challenges remain in determining the mechanism of formation of these superstructures and the environmental factors that can precisely modulate them. Notably the role that a functional ligand plays in the process of protein aggregation is largely unexplored. We herein address these issues with an original flavin-dependent RNA methyltransferase (TrmFO) used as a protein model since this protein employs a complex set of cofactors and ligands for catalysis. Here, we show that TrmFO carries an unstable protein structure that can partially mis-unfold leading to either formation of irregular and nonfunctional soluble oligomers endowed with hyper-thermal stability or large amorphous aggregates in the presence of salts. Mutagenesis confirmed that this peculiarity is an intrinsic property of a polypeptide and it is independent of the flavin coenzyme. Structural characterization and kinetic studies identified several regions of the protein that enjoy conformational changes and more particularly pinpointed the N-terminal subdomain as being a key element in the mechanisms of oligomerization and aggregation. Only stabilization of this region via tRNA suppresses these aberrant protein states. Although protein chaperones emerged as major actors against aggregation, our study emphasizes that other powerful mechanisms exist such as the stabilizing effect of functional assemblies that provide an additional layer of protection against the instability of the proteome.

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork.

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.

A two-component, multimeric endolysin encoded by a single gene.

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the conformation of the marginally stable LPPs to avoid their inappropriate protein/protein interactions and aggregation.

Biochemical and structural insights into microtubule perturbation by CopN from Chlamydia pneumoniae.

Although the actin network is commonly hijacked by pathogens, there are few reports of parasites targeting microtubules. The proposed member of the LcrE protein family from some Chlamydia species (e.g. pCopN from C. pneumoniae) binds tubulin and inhibits microtubule assembly in vitro. From the pCopN structure and its similarity with that of MxiC from Shigella, we definitively confirm CopN as the Chlamydia homolog of the LcrE family of bacterial proteins involved in the regulation of type III secretion. We have also investigated the molecular basis for the pCopN effect on microtubules. We show that pCopN delays microtubule nucleation and acts as a pure tubulin-sequestering protein at steady state. It targets the ß subunit interface involved in the tubulin longitudinal self-association in a way that inhibits nucleotide exchange. pCopN contains three repetitions of a helical motif flanked by disordered N- and C-terminal extensions. We have identified the pCopN minimal tubulin-binding region within the second and third repeats. Together with the intriguing observation that C. trachomatis CopN does not bind tubulin, our data support the notion that, in addition to the shared function of type III secretion regulation, these proteins have evolved different functions in the host cytosol. Our results provide a mechanistic framework for understanding the C. pneumoniae CopN-specific inhibition of microtubule assembly.

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

BACKGROUND: Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is "Generally Recognized As Safe". Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development. RESULTS: A recombinant ORF encoding a C-terminal, soluble, proteolytically inactive and tagged form of each staphylococcal HtrA protein was cloned into a lactococcal expression-secretion vector. After growth and induction of recombinant gene expression, L. lactis was able to produce and secrete each recombinant rHtrA protein as a stable form that accumulated in the culture medium in similar amounts as the naturally secreted endogenous protein, Usp45. L. lactis growth in fermenters, in particular in a rich optimized medium, led to higher yields for each rHtrA protein. Protein purification from the lactococcal culture medium was easily achieved in one step and allowed recovery of highly pure and stable proteins whose identity was confirmed by mass spectrometry. Although rHtrA proteins were monomeric, they displayed the same secondary structure content, thermal stability and chaperone activity as many other HtrA family members, indicating that they were correctly folded. rHtrA protein immunogenicity was established in mice. The raised polyclonal antibodies allowed studying the expression and subcellular localization of wild type proteins in S. aureus: although both proteins were expressed, only HtrA1 was found to be, as predicted, exposed at the staphylococcal cell surface suggesting that it could be a better candidate for vaccine development. CONCLUSIONS: In this study, an efficient process was developed to produce and secrete putative staphylococcal surface antigens in L. lactis and to purify them to homogeneity in one step from the culture supernatant. This allowed recovering fully folded, stable and pure proteins which constitute promising vaccine candidates to be tested for protection against staphylococcal infection. L. lactis thus proved to be an efficient and competitive cell factory to produce proteins of high quality for medical applications.

Development of monoclonal antibodies in tablet form: A new approach for local delivery.

Among the various pharmaceutical forms, tablets offer numerous advantages, like ease of administration, cost-effectiveness in production, and better stability of biomolecules. Beyond these benefits, the tablet form opens up possibilities for alternative routes for the local delivery of biopharmaceuticals such as oral or vaginal administration, thereby expanding the therapeutic applications of these biomolecules and overcoming the inconvenients associated with parenteral administration. However, to date there is limited information on the feasibility of developing biomolecules in the tablet form. In this study, we have evaluated the feasibility of developing monoclonal antibodies in the tablet form while preserving their biological properties. Different excipients and process parameters were studied to assess their impact on the antibody's integrity during tableting. ELISA results show that applying compression pressure up to 100 MPa is not detrimental to the antibody's binding properties when formulated from a lyophilized powder containing trehalose or sucrose as the major excipient. This observation was confirmed with SPR and ultracentrifugation experiments, which demonstrated that neither the binding affinity for both Fc and Fab antibody fragments nor its aggregation rate are affected by the tableting process. After compression, the tablets containing the antibodies have been shown to be stable for 6 months at room temperature.