Reduced Binding Affinity Research Articles

The base and root of domain II loops of Cry toxins contribute to binding to Bombyx mori ABC transporter C2.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.

Assessment of the binding interactions of SARS-CoV-2 spike glycoprotein variants

Severe acute respiratory syndrome-associated coronavirus 2 is a major global health issue and is driving the need for new therapeutics. The surface spike protein, which plays a central role in virus infection, is currently the target for vaccines and neutralizing treatments. The emergence of novel variants with multiple mutations in the spike protein may reduce the effectiveness of neutralizing antibodies by altering the binding activity of the protein with angiotensin-converting enzyme 2 (ACE2). To understand the impact of spike protein mutations on the binding interactions required for virus infection and the effectiveness of neutralizing monoclonal antibody (mAb) therapies, the binding activities of the original spike protein receptor binding domain (RBD) sequence and the reported spike protein variants were investigated using surface plasmon resonance. In addition, the interactions of the ACE2 receptor, an anti-spike mAb (mAb1), a neutralizing mAb (mAb2), the original spike RBD sequence, and mutants D614G, N501Y, N439K, Y453F, and E484K were assessed. Compared to the original RBD, the Y453F and N501Y mutants displayed a significant increase in ACE2 binding affinity, whereas D614G had a substantial reduction in binding affinity. All mAb-RBD mutant proteins displayed a reduction in binding affinities relative to the original RBD, except for the E484K-mAb1 interaction. The potential neutralizing capability of mAb1 and mAb2 was investigated. Accordingly, mAb1 failed to inhibit the ACE2-RBD interaction while mAb2 inhibited the ACE2-RBD interactions for all RBD mutants, except mutant E484K, which only displayed partial blocking.

An Attenuated Targeted-TNF Localizes to Tumors In Vivo and Regains Activity at the Site of Disease.

Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 μg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.

Peptide Nucleic Acid (PNA)-Guided Peptide Engineering of an Aptamer Sensor for Protease-Triggered Molecular Imaging.

Protease-triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnology. Herein, we report the design of a protease-activatable aptamer system that can perform molecular sensing and imaging in a tumor-specific manner. The system is constructed by locking the structure-switching activity of an aptamer using a rationally designed PNA-peptide-PNA triblock copolymer. Highly selective protease-mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell-selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.

Peptide Nucleic Acid (PNA)‐Guided Peptide Engineering of an Aptamer Sensor for Protease‐Triggered Molecular Imaging

AbstractProtease‐triggered control of functional DNA has remained unachieved, leaving a significant gap in activatable DNA biotechnology. Herein, we report the design of a protease‐activatable aptamer system that can perform molecular sensing and imaging in a tumor‐specific manner. The system is constructed by locking the structure‐switching activity of an aptamer using a rationally designed PNA–peptide–PNA triblock copolymer. Highly selective protease‐mediated cleavage of the peptide substrate results in reduced binding affinity of PNA to the aptamer module, with the subsequent recovery of its biosensing function. We demonstrated that the DNA/peptide/PNA hybrid system allows for tumor cell‐selective ATP imaging in vitro and also produces a fluorescent signal in vivo with improved tumor specificity. This work illustrates the potential of bridging the gap between functional DNA and peptides for precise biomedical applications.

Heparin promotes fibrillation of most phenol-soluble modulin virulence peptides from Staphylococcus aureus

Phenol-soluble modulins (PSMs), such as α-PSMs, β-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits β-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than β-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.

Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant

The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant.

Effects of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, cellular uptake, and gene expression

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5′-TACGAT-3′, which is found embedded on the 5′ flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.

Protein-imprinted polymer films prepared via cavity-selective multi-step post-imprinting modifications for highly selective protein recognition.

The use of molecularly imprinted polymers (MIPs) for achieving synthetic receptors capable of selective molecular recognition is promising; however, these polymers exhibit low selectivity derived from the heterogeneity of their created, imprinted cavities. To achieve highly selective protein recognition, we herein report the cavity-selective, multi-step, post-imprinting modification of MIPs. An MIP film for lysozyme was prepared by the copolymerization of {[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy acetic acid, a functional monomer possessing a modifiable disulfide bond, with acrylamide and N,N'-methylenebisacrylamide in the presence of lysozyme. After the removal of lysozyme, the disulfide bonds were cleaved by treatment with a reductant. A low concentration of lysozyme was then added to occupy the high-affinity cavities of the polymer and sterically protect the thiol groups within them. A poly(ethylene glycol)-based capping agent was reacted with the thiol groups residing in low-affinity cavities to hinder them. After the regeneration of the high-affinity cavities by washing out the bound lysozyme, the remaining thiol groups were reacted with 3-(2-pyridyldithio)propionic acid to introduce interacting groups, which produced capped MIPs. Comparing the capped and uncapped MIPs revealed that off-target protein binding was effectively suppressed by the capping treatment without any reduction in binding affinity (1.1 × 109M-1). Further investigation revealed that the lysozyme concentration during the capping process is critical for the selectivity of the capped MIP. In this case, highly selective MIPs were achieved when the lowest lysozyme concentration (100nM) was used. This facile process for creating highly selective, synthetic polymer receptors is a powerful approach for achieving plastic antibodies.

Flexible Loop in Carbohydrate-Binding Module 48 Allosterically Modulates Substrate Binding of the 1,4-α-Glucan Branching Enzyme.

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.

Mutations in the B.1.1.7 SARS-CoV-2 Spike Protein Reduce Receptor-Binding Affinity and Induce a Flexible Link to the Fusion Peptide.

The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. Amino acid exchanges within the SARS-CoV-2 spike protein variant of B.1.1.7 affect inter-monomeric contact sites within the trimer (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability or ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. Furthermore, we found increased flexibility in direct spatial proximity of the fusion peptide due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7. This study also implies a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD–ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2. Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD–ACE2 interface. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7.

An optimal antitumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers.

BackgroundCD3-based bispecific T cell engagers (bsTCEs) are one of the most promising bispecific antibodies for effective cancer treatments. To elicit target-specific T cell-mediated cytotoxicity, these bsTCEs contain at least one binding unit directed against a tumor antigen and another binding unit targeting CD3 in T cell receptor complex. Development of CD3-based bsTCEs, however, has been severely hampered by dose-limiting toxicities due to cytokine release syndrome. To address this limitation, we developed a novel functionally trivalent T cell engager (t-TCE) antibody containing affinity-reduced CD3 binding unit positioned to ensure monovalent CD3 engagement, in combination with bivalent tumor antigen binding of carcinoembryonic antigen (CEA).MethodsWe modeled the variable region of anti-CD3 in the complementarity-determining regions of the heavy chain and obtained CD3 binders with reduced binding affinity. Two optimized versions CEA/CD3-v1 and CEA/CD3-v2 were identified and generated in tetravalent format, characterized and compared in vitro and in vivo for functional activity.ResultsOur lead candidate, CEA/CD3-v2, demonstrated subnanomolar binding and picomolar potency against a panel of CEA-expressing cancer cell lines. In addition, we detected reduced T cell cytokine release with potent cytotoxic activity. Our t-TCE CEA/CD3-v2 molecule demonstrated strong antitumor effect in a dose-dependent manner in human peripheral blood mononuclear cell (PBMC) xenograft model. Furthermore, combination of CEA/CD3-v2 with atezolizumab provided synergistic antitumor effect.ConclusionsBecause of its effective tumor cell killing in vitro and in vivo with reduced cytokine release, CEA/CD3 bsTCE may greatly benefit in CEA-positive cancer immunotherapy.

Targeted design of drug binding sites in the main protease of SARS-CoV-2 reveals potential signatures of adaptation

Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (Mpro), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro. Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future.

The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication.

The duplication and ninefold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here, we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its subcellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an antiparallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable heterotrimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a sixteenfold reduced binding affinity for Gorab that cannot support centriole duplication. Thus, Gorab dimers at the Golgi exist in equilibrium with Sas6-associated monomers at the centriole to balance Gorab's dual role.

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2.

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.

Investigation on the binding interaction of rhodamine B with human serum albumin: effect of metal ions

The binding of rhodamine B (RB) to human serum albumin (HSA) in the absence and presence of Cu2+ or Fe3+ under simulated physiological conditions was studied by using various biophysical methods for the first time. The results showed that the interaction between HSA and RB could spontaneously result in the formation of HSA-RB complex (namely, static quenching mechanism) through hydrophobic interactions and hydrogen bonds irrespective of the absence or presence of metal ions. The presence of metal ions led to the reduction of binding affinity of RB to HSA compared with no metal ions, which might result from the conformational change of HSA caused by the binding of metal ions. Furthermore, the analysis of UV-vis absorption, circular dichroism, synchronous fluorescence and three-dimensional fluorescence experiments demonstrated that the addition of RB induced conformational and microenvironmental changes of HSA without and with metal ions. In short, this work will be helpful to in-depth understand the transport mechanism and biological effect of RB and the effect of metal ions on the interaction of HSA-RB in vivo.

The F19W mutation reduces the binding affinity of the transmembrane Aβ11-40 trimer to the membrane bilayer.

Alzheimer's disease is linked to the aggregation of the amyloid-β protein (Aβ) of 40 or 42 amino acids. Lipid membranes are known to modulate the rate and mechanisms of the Aβ aggregation. Point mutations in Aβ can alter these rates and mechanisms. In particular, experiments show that F19 mutations influence the aggregation rate, but maintain the fibril structures. Here, we used molecular dynamics simulations to examine the effect of the F19W mutation in the 3Aβ11–40 trimer immersed in DPPC lipid bilayers submerged in aqueous solution. Substituting Phe by its closest (non-polar) aromatic amino acid Trp has a dramatic reduction in binding affinity to the phospholipid membrane (measured with respect to the solvated protein) compared to the wild type: the binding free energy of the protein–DPPC lipid bilayer increases by 40–50 kcal mol−1 over the wild-type. This is accompanied by conformational changes and loss of salt bridges, as well as a more complex free energy surface, all indicative of a more flexible and less stable mutated trimer. These results suggest that the impact of mutations can be assessed, at least partially, by evaluating the interaction of the mutated peptides with the lipid membranes.

Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting

The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy.

Enzyme kinetic and binding studies identify determinants of specificity for the immunomodulatory enzyme ScpA, a C5a inactivating bacterial protease

The Streptococcal C5a peptidase (ScpA) specifically inactivates the human complement factor hC5a, a potent anaphylatoxin recently identified as a therapeutic target for treatment of COVID-19 infections. Biologics used to modulate hC5a are predominantly monoclonal antibodies. Here we present data to support an alternative therapeutic approach based on the specific inactivation of hC5a by ScpA in studies using recombinant hC5a (rhC5a). Initial characterization of ScpA confirmed activity in human serum and against rhC5a desArg (rhC5adR), the predominant hC5a form in blood. A new FRET based enzyme assay showed that ScpA cleaved rhC5a at near physiological concentrations (Km 185 nM). Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) studies established a high affinity ScpA-rhC5a interaction (KD 34 nM, KDITC 30.8 nM). SPR analyses also showed that substrate binding is dominated (88% of ΔG°bind) by interactions with the bulky N-ter cleavage product (PN, ’core’ residues 1–67) with interactions involving the C-ter R74 contributing most of the remaining ΔG°bind. Furthermore, reduced binding affinity following mutation of a subset of positively charged Arginine residues of PN and in the presence of higher salt concentrations, highlighted the importance of electrostatic interactions. These data provide the first in-depth study of the ScpA-C5a interaction and indicate that ScpA’s ability to efficiently cleave physiological concentrations of C5a is driven by electrostatic interactions between an exosite on the enzyme and the ‘core’ of C5a. The results and methods described herein will facilitate engineering of ScpA to enhance its potential as a therapeutic for excessive immune response to infectious disease.

Mechanistic insights into the effect of phosphorylation on Ras conformational dynamics and its interactions with cell signaling proteins

Ras undergoes interconversion between the active GTP-bound state and the inactive GDP-bound state. This GTPase cycle, which controls the activities of Ras, is accelerated by Ras GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (SOS). Oncogenic Ras mutations could affect the GTPase cycle and impair Ras functions. Additionally, Src-induced K-Ras Y32/64 dual phosphorylation has been reported to disrupt GTPase cycle and hinder Ras downstream signaling. However, the underlying mechanisms remain unclear. To address this, we performed molecular dynamics simulations (~30 μs in total) on unphosphorylated and phosphorylated K-Ras4B in GTP- and GDP-bound states, and on their complexes with GTPase cycle regulators (GAP and SOS) and the effector protein Raf. We found that K-Ras4B dual phosphorylation mainly alters the conformation at the nucleotide binding site and creates disorder at the catalytic site, resulting in the enlargement of GDP binding pocket and the retard of Ras-GTP intrinsic hydrolysis. We observed phosphorylation-induced shift in the distribution of Ras-GTP inactive-active sub-states and recognized potential druggable pockets in the phosphorylated Ras-GTP. Moreover, decreased catalytic competence or signal delivery abilities due to reduced binding affinities and/or distorted catalytic conformations of GAP, SOS and Raf were observed. In addition, the allosteric pathway from Ras/Raf interface to the distal Raf L4 loop was compromised by Ras phosphorylation. These results reveal the mechanisms by which phosphorylation influences the intrinsic or GAP/SOS catalyzed transformations between GTP- and GDP-bound states of Ras and its signal transduction to Raf. Our findings project Ras phosphorylation as a target for cancer drug discovery.