We present a possible molecular basis for the opposite activity of two homologues proteins that bind similar ligands and show that this is achieved by fine-tuning of the interaction interface. The highly homologous ASPP proteins have opposite roles in regulating apoptosis: ASPP2 induces apoptosis while iASPP inhibits it. The ASPP proteins are regulated by an autoinhibitory interaction between their Ank-SH3 and Pro domains. We performed a detailed biophysical and molecular study of the Pro – Ank-SH3 interaction in iASPP and compared it to the interaction in ASPP2. We found that iASPP Pro is disordered and that the interaction sites are entirely different: iASPP Ank-SH3 binds iASPP Pro via its fourth Ank repeat and RT loop while ASPP2 Ank-SH3 binds ASPP2 Pro via its first Ank repeat and the n-src loop. It is possible that by using different moieties in the same interface, the proteins can have distinct and specific interactions resulting in differential regulation and ultimately different biological activities.

We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. Our approach combines phosphoproteomics for high throughput cancer-related biomarker discovery from patient tissues and an impedimetric kinase activity biosensor for sensitive validation. Using non-small-cell lung cancer (NSCLC) as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed 198 phosphoproteins that are over-phosphorylated in NSCLC. Among the differentially regulated phosphorylation sites, the most significant alteration was in residue S165 in the Hepatoma Derived Growth Factor (HDGF) protein. Hence, HDGF was selected as a model system for the electrochemical studies. Further motif-based analysis of this altered phosphorylation site revealed that extracellular-signal-regulated kinase 1/2 (ERK1/2) are most likely to be the corresponding kinases. For validation of the kinase–substrate pair, densely packed peptide monolayers corresponding to the HDGF phosphorylation site were coupled to a gold electrode. Phosphorylation of the monolayer by ERK2 and dephosphorylation by alkaline phosphatase (AP) were detected by electrochemical impedance spectroscopy (EIS) and surface roughness analysis. Compared to other methods for quantification of kinase concentration, this label-free electrochemical assay offers the advantages of ultra-sensitivity as well as higher specificity for the detection of cancer-related kinase–substrate pair. With implementation of multiple kinase–substrate biomarker pairs, we expect this integrated approach to become a high throughput platform for discovery and validation of phosphorylation-mediated biomarkers.

The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express and translocate the oncoprotein CagA into host epithelial cells. Once inside host cells, CagA interacts with ASPP2, which specifically stimulates p53-mediated apoptosis and reverses its pro-apoptotic function to promote ASPP2-dependent degradation of p53. The X-ray crystal structure of a complex between the N-terminal domain of CagA and a 56-residue fragment of ASPP2, of which 22 residues were resolved, was recently described. Here, we present biochemical and biophysical analyses of the interaction between the additional regions of CagA and ASPP2 potentially involved in this interaction. Using size exclusion chromatography–multiangle laser light scattering, circular dichroism, and nuclear magnetic resonance analyses, we observed that the ASPP2 region spanning residues 331–692, which was not part of the ASPP2 fragment used for crystallization, is intrinsically disordered in its unbound state. By surface plasmon resonance analysis and isothermal titration calorimetry, we found that a portion of this disordered region in ASPP2, residues 448–692, binds to the N-terminal domain of CagA. We also measured the affinity of the complex between the ASPP2 fragment composed of residues 693–918 and inclusive of the fragment used for crystallization and CagA. Additionally, we mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating interactions between CagA and numerous peptides distributed throughout the ASPP2 protein sequence. Our results identify previously uncharacterized regions distributed throughout the protein sequence of ASPP2 as determinants of CagA binding, providing mechanistic insight into apoptosis reprogramming by CagA and potential new drug targets for H. pylori-mediated gastric cancer.

We have previously introduced an easy to perform, cost-effective and highly efficient acetylation technique for solid phase synthesis (SPPS). Malonic acid is used as a precursor and the reaction proceeds via a reactive ketene that acetylates the target amine. Here we present a detailed mechanistic study of the malonic acid-mediated acylation. The influence of reaction conditions, peptide sequence and reagents was systematically studied. Our results show that the methodology can be successfully applied to different types of peptides and nonpeptidic molecules irrespective of their structure, sequence, or conformation. Using alkyl, phenyl, and benzyl malonic acid, we synthesized various acyl peptides with almost quantitative yields. The ketenes obtained from the different malonic acid derived precursors were characterized by in situ (1) H-NMR. The reaction proceeded in short reaction times and resulted in excellent yields when using uronium-based coupling agents, DIPEA as a base, DMF/DMSO/NMP as solvents, Rink amide/Wang/Merrifield resins, temperature of 20°C, pH 8-12 and 5 min preactivation at inert atmosphere. The reaction was unaffected by Lewis acids, transition metal ions, surfactants, or salt. DFT studies support the kinetically favorable concerted mechanism for CO2 and ketene formation that leads to the thermodynamically stable acylated products. We conclude that the malonic acid-mediated acylation is a general method applicable to various target molecules.

Many proteins are prone to aggregate or insoluble for different reasons. This poses an extraordinary challenge at the expression level, but even more during downstream purification processes. Here we describe a strategy that we developed for purifying prone-to-aggregate proteins. Our methodology can be easily implemented in small laboratories without the need for automated, expensive platforms. This procedure is especially suitable for intrinsically disordered proteins (IDPs) and for proteins with intrinsically disordered regions (IDRs). Such proteins are likely to aggregate due to their lack of tertiary structure and their extended and flexible conformations. Similar methodologies can be applied to other proteins with comparable tendency to aggregate during the expression or purification steps. In this chapter, we will mainly focus on protein solubility and stability issues during purification and storage, on factors that can prevent aggregation or maintain solubility, and on the importance of the early elimination of aggregates during protein purification.

Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the “shiftides” approach developed in our lab.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium Magnetospirillum gryphiswaldense as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the M. gryphiswaldense MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested in vivo. Additionally, by combining several in vitro and in silico methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDF's were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes – bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domain's conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

Life requires orchestrated control of cell proliferation, cell maintenance, and cell death. Involved in these decisions are protein complexes that assimilate a variety of inputs that report on the status of the cell and lead to an output response. Among the proteins involved in this response are nutrient-deprivation autophagy factor-1 (NAF-1)- and Bcl-2. NAF-1 is a homodimeric member of the novel Fe-S protein NEET family, which binds two 2Fe-2S clusters. NAF-1 is an important partner for Bcl-2 at the endoplasmic reticulum to functionally antagonize Beclin 1-dependent autophagy [Chang NC, Nguyen M, Germain M, Shore GC (2010) EMBO J 29(3):606–618]. We used an integrated approach involving peptide array, deuterium exchange mass spectrometry (DXMS), and functional studies aided by the power of sufficient constraints from direct coupling analysis (DCA) to determine the dominant docked conformation of the NAF-1–Bcl-2 complex. NAF-1 binds to both the pro- and antiapoptotic regions (BH3 and BH4) of Bcl-2, as demonstrated by a nested protein fragment analysis in a peptide array and DXMS analysis. A combination of the solution studies together with a new application of DCA to the eukaryotic proteins NAF-1 and Bcl-2 provided sufficient constraints at amino acid resolution to predict the interaction surfaces and orientation of the protein–protein interactions involved in the docked structure. The specific integrated approach described in this paper provides the first structural information, to our knowledge, for future targeting of the NAF-1–Bcl-2 complex in the regulation of apoptosis/autophagy in cancer biology.

Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable His-tagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.

We describe a new general N-acetylation method for solid phase synthesis. Malonic acid is used as a precursor and the reaction proceeds by in situ formation of a reactive ketene intermediate at room temperature. We have successfully applied this methodology to peptides and non-peptidic molecules containing a variety of functional groups. The reaction gave high yields compared to known acetylation methods, irrespective of the structure, conformation and sequence of the acetylated molecule. Computational studies revealed that the concerted mechanism via the ketene intermediate is kinetically favorable and leads to a thermodynamically stable acetylated product. In conclusion, our method can be easily applied to acetylation in a wide variety of chemical reactions performed on the solid phase.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.

In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

The pro-apoptotic ASPP2 protein plays a central role in regulating apoptosis in both p53-dependent and p53-independent pathways. It has become clear in recent years that ASPP2 plays also a role in other important cellular processes like senescence and regulating cell polarity. ASPP2 interacts with numerous proteins in order to exert its pro-apoptotic effect while some other proteins inhibit its pro-apoptotic activity. These interactions are emerging potential targets for activation or inhibition. Drugs that activate ASPP2 may not only induce apoptosis of cancer cells, but also affect other disease-related cellular pathways. Here we review the interactions of ASPP2 with its partner proteins and their potential targeting for drug development.

Intrinsically disordered proteins (IDPs) or regions (IDRs) in proteins hold many functions but their biological roles are still not fully understood. Here we describe a new role of such regions. Using the HIV-1 Rev protein, we show that disordered domains have a role in maintaining the correct oligomeric state and the thermodynamic stability of proteins.

We present a new approach for peptide cyclization during solid phase synthesis under highly acidic conditions. Our approach involves simultaneous in situ deprotection, cyclization and trifluoroacetic acid (TFA) cleavage of the peptide, which is achieved by forming an amide bond between a lysine side chain and a succinic acid linker at the peptide N-terminus. The reaction proceeds via a highly active succinimide intermediate, which was isolated and characterized. The structure of a model cyclic peptide was solved by NMR spectroscopy. Theoretical calculations support the proposed mechanism of cyclization. Our new methodology is applicable for the formation of macrocycles in solid-phase synthesis of peptides and organic molecules.

Occurrence of DNA damage in a cell activates the DNA damage response, a survival mechanism that ensures genomics stability. Two key members of the DNA damage response are the tumor suppressor p53, which is the most frequently mutated gene in cancers, and MDC1, which is a central adaptor that recruits many proteins to sites of DNA damage. Here we characterize the in vitro interaction between p53 and MDC1 and demonstrate that p53 and MDC1 directly interact. The p53-MDC1 interaction is mediated by the tandem BRCT domain of MDC1 and the C-terminal domain of p53. We further show that both acetylation of lysine 382 and phosphorylation of serine 392 in p53 enhance the interaction between p53 and MDC1. Additionally, we demonstrate that the p53-MDC1 interaction is augmented upon the induction of DNA damage in human cells. Our data suggests a new role for acetylation of lysine 382 and phosphorylation of serine 392 in p53 in the cellular stress response and offers the first evidence for an interaction involving MDC1 that is modulated by acetylation.

The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr¹⁹⁵⁷ or Thr¹⁹⁶⁰ inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.

Integration of the HIV cDNA into the host chromosome is a key event in the viral replication cycle. It is mediated by the viral integrase (IN) enzyme, which is an attractive anti-HIV drug target. Here we present the first AFM imaging of IN-mediated DNA integration products in a two-LTR system.