Bence Kiss

Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4–NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4–NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca2+-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4–NMIIA interaction.

MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with Kd values of 9 and 40 μm, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: Kd values of 37 and 3.5 nm for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent Kd value (3 μm). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network.

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca2+-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca2+-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a “fuzzy” complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

Phosphorylation as conformational switch from the native to amyloid state: Trp-cage as a protein aggregation model

The 20 residue long Trp-cage miniprotein is an excellent model for both computational and experimental studies of protein folding and stability. Recently, great attention emerged to study disease-related protein misfolding, aggregation, and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects. It aggregates with ease upon structure destabilization, and thus it is suitable for modeling aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, molecular dynamics (MD) simulations, and transmission electron microscopy (TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant, which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form that can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.

Structural determinants governing S100A4‐induced isoform‐selective disassembly of nonmuscle myosin II filaments

The Ca2+‐binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA (NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 (NMIIA numbering), the NMIIA/NMIIC‐specific alanine provides about 60‐fold higher affinity than the NMIIB‐specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α‐helical binding segments. This communication is completely abolished by the Ala‐to‐Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C‐terminal coiled‐coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform‐selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4‐dependent and ‐independent regulations, respectively.

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2.

Transglutaminase-2 (TG2) is best known as a Ca(2+)-dependent cross-linking enzyme; however, some of its extracellular matrix-related functions are independent of its catalytic activity and include matrix remodelling, adhesion and migration. S100A4 belongs to the Ca(2+)-binding EF-hand S100 protein family and acts both intra- and extra-cellularly through binding to various partners. It regulates cell migration and its overexpression is strongly associated with metastasis and poor survival in various cancers. It has recently been suggested that TG2 mediates S100A4-dependent tumour cell migration. In the present study we provide evidence that S100A4 is an interacting partner and also a specific amine donor of TG2. TG2 incorporates a glutamine donor peptide to Lys(100) in the C-terminal random coil region of S100A4. Importantly, the enzyme activity is not necessary for the interaction: S100A4 also binds to TG2 in the presence of a specific inhibitor that keeps the enzyme in an open conformation, or to an enzymatically inactive mutant. We also found that S100A4 considerably enhances TG2-mediated adhesion of A431 epithelial carcinoma cells to the extracellular matrix. This role is independent of enzyme activity and requires the open conformation of TG2. We propose that S100A4 stabilizes the open conformation of TG2, which binds to its cell-surface receptor in this state and increases cell adhesion.

The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation

S100A4 interacts with many binding partners upon Ca2+ activation and is strongly associated with increased metastasis formation. In order to understand the role of the C-terminal random coil for the protein function we examined how small angle X-ray scattering of the wild-type S100A4 and its C-terminal deletion mutant (residues 1–88, Δ13) changes upon Ca2+ binding. We found that the scattering intensity of wild-type S100A4 changes substantially in the 0.15–0.25 Å−1 q-range whereas a similar change is not visible in the C-terminus deleted mutant. Ensemble optimization SAXS modeling indicates that the entire C-terminus is extended when Ca2+ is bound. Pulsed field gradient NMR measurements provide further support as the hydrodynamic radius in the wild-type protein increases upon Ca2+ binding while the radius of Δ13 mutant does not change. Molecular dynamics simulations provide a rational explanation of the structural transition: the positively charged C-terminal residues associate with the negatively charged residues of the Ca2+-free EF-hands and these interactions loosen up considerably upon Ca2+-binding. As a consequence the Δ13 mutant has increased Ca2+ affinity and is constantly loaded at Ca2+ concentration ranges typically present in cells. The activation of the entire C-terminal random coil may play a role in mediating interaction with selected partner proteins of S100A4.

Novel linear motif filtering protocol reveals the role of the LC8 dynein light chain in the Hippo pathway

Protein-protein interactions (PPIs) formed between short linear motifs and globular domains play important roles in many regulatory and signaling processes but are highly underrepresented in current protein-protein interaction databases. These types of interactions are usually characterized by a specific binding motif that captures the key amino acids shared among the interaction partners. However, the computational proteome-level identification of interaction partners based on the known motif is hindered by the huge number of randomly occurring matches from which biologically relevant motif hits need to be extracted. In this work, we established a novel bioinformatic filtering protocol to efficiently explore interaction network of a hub protein. We introduced a novel measure that enabled the optimization of the elements and parameter settings of the pipeline which was built from multiple sequence-based prediction methods. In addition, data collected from PPI databases and evolutionary analyses were also incorporated to further increase the biological relevance of the identified motif hits. The approach was applied to the dynein light chain LC8, a ubiquitous eukaryotic hub protein that has been suggested to be involved in motor-related functions as well as promoting the dimerization of various proteins by recognizing linear motifs in its partners. From the list of putative binding motifs collected by our protocol, several novel peptides were experimentally verified to bind LC8. Altogether 71 potential new motif instances were identified. The expanded list of LC8 binding partners revealed the evolutionary plasticity of binding partners despite the highly conserved binding interface. In addition, it also highlighted a novel, conserved function of LC8 in the upstream regulation of the Hippo signaling pathway. Beyond the LC8 system, our work also provides general guidelines that can be applied to explore the interaction network of other linear motif binding proteins or protein domains.

Multilevel Changes in Protein Dynamics upon Complex Formation of the Calcium-Loaded S100A4 with a Nonmuscle Myosin IIA Tail Fragment

Dysregulation of Ca2+‐binding S100 proteins plays important role in various diseases. The asymmetric complex of Ca2+‐bound S100A4 with nonmuscle myosin IIA has high stability and highly increased Ca2+ affinity. Here we investigated the possible causes of this allosteric effect by NMR spectroscopy. Chemical shift‐based secondary‐structure analysis did not show substantial changes for the complex. Backbone dynamics revealed slow‐timescale local motions in the H1 helices of homodimeric S100A4; these were less pronounced in the complex form and might be accompanied by an increase in dimer stability. Different mobilities in the Ca2+‐coordinating EF‐hand sites indicate that they communicate by an allosteric mechanism operating through changes in protein dynamics; this must be responsible for the elevated Ca2+ affinity. These multilevel changes in protein dynamics as conformational adaptation allow S100A4 fine‐tuning of its protein–protein interactions inside the cell during Ca2+ signaling.