Structural Complementation of the Catalytic Domain of Pseudomonas Exotoxin A

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.     

Enhancement of Cry19Aa Mosquitocidal Activity against Aedes aegypti by Mutations in the Putative Loop Regions of Domain II

Improvements in the mosquitocidal activity of Bacillus thuringiensis Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of Aedes toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against Anopheles or Culex.     

A Combined Quantum/Classical Molecular Dynamics Study of the Catalytic Mechanism of HIV Protease

Based on available three-dimensional structures of enzyme-inhibitor complexes, the mechanism of the reaction catalysed by HIV protease is studied using molecular dynamics simulations with molecular mechanics and combined quantum-mechanics/molecular-mechanics potential energy functions. The results support the general acid/general base catalysis mechanism, with Asp25′ protonated in the enzyme-substrate complex. In the enzyme-substrate complex, the lytic water molecule binds at a position different from the positions of the hydroxyl groups in various aspartic protease-inhibitor complexes. The carboxyl groups at the active site also adopt a different orientation. However, when the lytic water molecule approaches the scissile peptide, the reaction centre changes gradually to a conformation close to that derived from X-ray diffraction studies of various enzyme-inhibitor complexes. The proton transfer processes can take place only after the lytic water molecule has approached the scissile peptide bond to a certain degree. Qualitatively, the free-energy barrier associated with the nucleophilic attack step, which takes place at physiological pH, is comparable with the acid or base-catalysed reactions of model systems. The structure of the tetrahedral intermediate resulting from the nucleophilic attack step also indicates a straightforward pathway of the next reaction step, i.e. the breaking of the C-N bond.     

Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae

Linalool production was evaluated in different Saccharomyces cerevisiae strains expressing the Clarkia breweri linalool synthase gene (LIS). The wine strain T₇₃ was shown to produce higher levels of linalool than conventional laboratory strains (i.e., almost three times the amount). The performance of this strain was further enhanced by manipulating the endogenous mevalonate (MVA) pathway: deregulated overexpression of the rate-limiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) doubled linalool production. In a haploid laboratory strain, engineering of this key step also improved linalool yield.Monoterpenes are a class of isoprenoids of increasing industrial and clinical interest usually produced by plants. They are used as aromatic additives in the food and cosmetics industries and are also important components in wine aroma. Moreover, certain monoterpenes display antimicrobial, antiparasitic, and antiviral properties as well as a plethora of promising health benefits (for recent reviews, see references 2, 7, 15, 28, and 30 and references cited therein). To date, many studies have focused on plant metabolic engineering of monoterpene production (for selected reviews, see references 1, 14, 19, 29, and 35 and references cited therein), and few studies have been carried out on microorganisms (9, 21, 22, 34, 38). Efficient microbial production of these metabolites could provide an alternative to the current methods of chemical synthesis or extraction from natural sources. In this regard, a considerable number of studies have shown the utility of Saccharomyces cerevisiae as a valuable platform for sesquiterpene, diterpene, triterpene, and carotene production (references 5, 10, 23, 26, 30, 31, 32, and 33 and references cited therein). However, all the efforts dedicated to the improvement of isoprenoid yields in S. cerevisiae have been performed using conventional laboratory strains, and there are no studies concerning natural or industrially relevant isolates.In recent years, many genes that encode plant monoterpene synthases (MTS), a family of enzymes which specifically catalyze the conversion of the ubiquitous C₁₀ intermediate of isoprenoid biosynthesis geranyl pyrophosphate (GPP) to monoterpenes, have been characterized. Such is the case with the LIS gene (codes for S-linalool synthase) of Clarkia breweri, the first MTS-encoding gene to be isolated (13). In contrast to plants, S. cerevisiae cannot produce monoterpenes efficiently, mainly due to the lack of specific pathways involving MTS. However, GPP is formed as a transitory intermediate in the two-step synthesis of farnesyl pyrophosphate (FPP), catalyzed by FPP synthase (FPPS) (Fig. ​(Fig.1),1), and some natural S. cerevisiae strains have been shown to possess the ability to produce small amounts of monoterpenes (8). Whether this occurs through unspecific dephosphorylation of a more available endogenous pool of GPP and subsequent bioconversions is not known. In addition, it has recently been established that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes under laboratory and vinification conditions (22, 34).Open in a separate windowFIG. 1.Simplified isoprenoid pathway in S. cerevisiae, including the branch point to linalool. Dotted arrows indicate that more than one reaction is required to convert the substrate to the product indicated. Dashed arrows indicate the engineered steps. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; HMGR, HMG-CoA reductase; FPPS, FPP synthase; LIS, linalool synthase.Here we present the process for selecting and optimizing yeast strains for foreign monoterpene production. We have chosen the C. breweri LIS gene as a prototype because, when heterologously expressed in S. cerevisiae, it specifically results in the production of linalool (3,7-dimethyl-1,6-octadien-3-ol; a floral scent and bioactive acyclic monoterpene identified in numerous fruits and flowers) and no other by-products (22). Two S. cerevisiae strains of different origins have been selected and their endogenous mevalonate (MVA) pathways engineered to enhance the production of linalool. These strategies might be employed to produce any other recombinant monoterpene in S. cerevisiae by expressing the appropriate monoterpene synthase.     

The Protease Domain Increases the Translocation Stepping Efficiency of the Hepatitis C Virus NS3-4A Helicase

Hepatitis C virus (HCV) NS3 protein has two enzymatic activities of helicase and protease that are essential for viral replication. The helicase separates the strands of DNA and RNA duplexes using the energy from ATP hydrolysis. To understand how ATP hydrolysis is coupled to helicase movement, we measured the single turnover helicase translocation-dissociation kinetics and the pre-steady-state P_i release kinetics on single-stranded RNA and DNA substrates of different lengths. The parameters of stepping were determined from global fitting of the two types of kinetic measurements into a computational model that describes translocation as a sequence of coupled hydrolysis-stepping reactions. Our results show that the HCV helicase moves with a faster rate on single stranded RNA than on DNA. The HCV helicase steps on the RNA or DNA one nucleotide at a time, and due to imperfect coupling, not every ATP hydrolysis event produces a successful step. Comparison of the helicase domain (NS3h) with the protease-helicase (NS3-4A) shows that the most significant contribution of the protease domain is to improve the translocation stepping efficiency of the helicase. Whereas for NS3h, only 20% of the hydrolysis events result in translocation, the coupling for NS3-4A is near-perfect 93%. The presence of the protease domain also significantly reduces the stepping rate, but it doubles the processivity. These effects of the protease domain on the helicase can be explained by an improved allosteric cross-talk between the ATP- and nucleic acid-binding sites achieved by the overall stabilization of the helicase domain structure.     

Cleavage of SNAP25 and Its Shorter Versions by the Protease Domain of Serotype A Botulinum Neurotoxin

Various substrates, catalysts, and assay methods are currently used to screen inhibitors for their effect on the proteolytic activity of botulinum neurotoxin. As a result, significant variation exists in the reported results. Recently, we found that one source of variation was the use of various catalysts, and have therefore evaluated its three forms. In this paper, we characterize three substrates under near uniform reaction conditions using the most active catalytic form of the toxin. Bovine serum albumin at varying optimum concentrations stimulated enzymatic activity with all three substrates. Sodium chloride had a stimulating effect on the full length synaptosomal-associated protein of 25 kDa (SNAP25) and its 66-mer substrates but had an inhibitory effect on the 17-mer substrate. We found that under optimum conditions, full length SNAP25 was a better substrate than its shorter 66-mer or 17-mer forms both in terms of k_cat, K_m, and catalytic efficiency k_cat/K_m. Assay times greater than 15 min introduced large variations and significantly reduced the catalytic efficiency. In addition to characterizing the three substrates, our results identify potential sources of variations in previous published results, and underscore the importance of using well-defined reaction components and assay conditions.     

Functional consequences of alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal part of transmembrane helix M3 of the Na+,K(+)-ATPase

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.     

The Impact of Individual Human Immunodeficiency Virus Type 1 Protease Mutations on Drug Susceptibility Is Highly Influenced by Complex Interactions with the Background Protease Sequence

The requirement for multiple mutations for protease inhibitor (PI) resistance necessitates a better understanding of the molecular basis of resistance development. The novel bioinformatics resistance determination approach presented here elaborates on genetic profiles observed in clinical human immunodeficiency virus type 1 (HIV-1) isolates. Synthetic protease sequences were cloned in a wild-type HIV-1 background to generate a large number of close variants, covering 69 mutation clusters between multi-PI-resistant viruses and their corresponding genetically closely related, but PI-susceptible, counterparts. The vast number of mutants generated facilitates a profound and broad analysis of the influence of the background on the effect of individual PI resistance-associated mutations (PI-RAMs) on PI susceptibility. Within a set of viruses, all PI-RAMs that differed between susceptible and resistant viruses were varied while maintaining the background sequence from the resistant virus. The PI darunavir was used to evaluate PI susceptibility. Single sets allowed delineation of the impact of individual mutations on PI susceptibility, as well as the influence of PI-RAMs on one another. Comparing across sets, it could be inferred how the background influenced the interaction between two mutations, in some cases even changing antagonistic relationships into synergistic ones or vice versa. The approach elaborates on patient data and demonstrates how the specific mutational background greatly influences the impact of individual mutations on PI susceptibility in clinical patterns.The clinical use of protease inhibitors (PIs) for the treatment of human immunodeficiency virus (HIV) infection has led to a remarkable decline in HIV-1-related morbidity and mortality, and PIs are now a cornerstone of highly active antiretroviral therapy (14). However, the clinical benefit of PIs is limited by several factors, including long-term safety and tolerability, resistance development, and drug-drug interactions.The combination of extremely high levels of virus production and a high mutation rate is resulting in a growing resistance to anti-HIV drugs, making these less effective over time (1). In addition, an increasing proportion of primary infections involve the transmission of resistant viruses, including strains with reduced susceptibility to approved PIs (17). Therefore, patients need to be monitored for development of drug resistance, and treatment regimens have to be adapted accordingly. Most currently approved PIs are based on similar chemical structures, and therefore extensive cross-resistance can occur (7).In order to investigate the molecular basis of resistance development, we used the PI darunavir (DRV) as a model. DRV, previously known as TMC114, was approved in 2006 for the treatment of highly experienced patients and in 2008 for treatment of naïve patients. DRV has a high in vitro and in vivo potency against wild-type (WT) HIV, and this activity is maintained against HIV variants that are highly cross-resistant to other licensed PIs (2, 15). Moreover, there appears to be a very high genetic barrier to the development of resistance to DRV (3). A diminished virological response to DRV was only observed at week 24 (POWER studies [4]), when at least three specific baseline protease mutations (of V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V, and L89V) occurred in a background containing multiple protease mutations (median of at least 10 International AIDS Society-USA [IAS-USA] PI resistance-associated mutations [PI-RAMs] [11]).Mutations can interact as part of higher-order networks in complex and frequently overlapping patterns (7, 16, 18). In such patterns, the effect of an individual protease mutation on drug susceptibility depends on the presence of other mutations, PI-RAMs as well as background mutations. Many of the background mutations act synergistically with PI-RAMs and increase resistance to specific drugs. In addition, some of these mutations favor the development of other drug resistance mutations, thus lowering the genetic barrier to the development of PI resistance. In contrast, some mutations in the mutational background antagonize the effects of an individual PI-RAM. As resistance mutations are usually associated with reduced viral fitness, it may be that certain background mutations could (partly) compensate for this (12).In order to design drugs with high genetic barriers to resistance, a full understanding of the molecular basis of resistance development is needed. This includes the complex interplay between resistance mutations that can be studied only by exploring genetically close variants. Because of the high variability of HIV, it is difficult to find the genetically related variants required for such a study in patient databases, even if they contain sequences from thousands of virus isolates. Traditional approaches utilizing site-directed mutagenesis to create close variants by modifying the protease amino acids in existing viruses are feasible only on a small scale. The advent of mature gene assembly technologies makes the large-scale generation of closely related variants practicable. Here we describe a novel approach, bioinformatics resistance determination (BIRD), in which we created PI resistance sets between viral genotypes observed in patient samples. By varying a specific set of mutations in an invariable genetic background, the complex interactions between these mutations could be carefully dissected. Our studies illustrate how some mutations do not influence other mutations, while other changes act synergistically or antagonistically toward a specific RAM. Moreover, by comparing sets, we show how a specific background can alter the interplay between mutations.     

Molecular Dynamics Simulation Reveals Insights into the Mechanism of Unfolding by the A130T/V Mutations within the MID1 Zinc-Binding Bbox1 Domain

The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β) and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD) simulation for the A130V mutant (>6 Å) and after 30 ns for A130T mutant (>6 Å). Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.     

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

Enterovirus A71(EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease(HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43(TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis(ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3 C between the residues 331 Q and332 S, while mutated TDP-43(Q331 A) was not cleaved. In addition, mutated 3 C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2 A rather than 3 C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71 infection.     

Road Map for the Structure-Based Design of Selective Covalent HCV NS3/4A Protease Inhibitors

Over the last 2 decades, covalent inhibitors have gained much popularity and is living up to its reputation as a powerful tool in drug discovery. Covalent inhibitors possess many significant advantages including increased biochemical efficiency, prolonged duration and the ability to target shallow, solvent exposed substrate-binding domains. However, rapidly mounting concerns over the potential toxicity, highly reactive nature and general lack of selectivity have negatively impacted covalent inhibitor development. Recently, a great deal of emphasis by the pharmaceutical industry has been placed toward the development of novel approaches to alleviate the major challenges experienced through covalent inhibition. This has unexpectedly led to the emergence of “selective” covalent inhibitors. The purpose of this review is not only to provide an overview from literature but to introduce a technical guidance as to how to initiate a systematic “road map” for the design of selective covalent inhibitors which we believe may assist in the design and development of optimized potential selective covalent HCV NS3/4A viral protease inhibitors.     

Enhancement of peptidyl transferase activity by antibiotics acting on the 50 S ribosomal subunit

Interconversions of ribosomes, between forms that are active and inactive in peptidyl transfer, were studied and conditions favoring a state of equilibrium between the two forms were established. Under such conditions activity was enhanced two-to fivefold by the antibiotics erythromycin, vernamycin Bα, lincomycin, chloramphenicol and vernamycin A. The antibiotics puromycin, gougerotin, thiostrepton and siomycin, whose target site is also the 50 S ribosomal subunit, were ineffective.A common feature of the effective antibiotics is their ability to bind to ribosomes active in peptidyl transfer but not to enzymatically inactive ribosomes. The activity enhancing effect of antibiotics is therefore interpreted as being due to a shift in the equilibrium between the two ribosomal forms in favor of the active conformation, brought about by the preferential binding of the antibiotic to ribosomes in this form. The results stress the flexible nature of ribosome structure and suggest that antibiotics can function as allosteric effectors in modifying ribosome conformation.     

泛素活化酶Ube1活性中心疏水区关键苯丙氨酸位点的丙氨酸突变对泛素传递活性的影响

泛素化系统调节真核细胞中几乎所有的生命活动,参与细胞内绝大多数蛋白质的降解,而疾病的发生往往伴随着相关蛋白质的异常。研究与疾病相关蛋白质的泛素化途径,通过该途径促进或抑制其活性对疾病的研究和治疗具有重大的意义。然而,细胞内的泛素传递网络错综复杂,天然条件下很难确认某一特定蛋白质的泛素化途径。我们前期工作建立的正交泛素传递途径中的泛素和泛素化酶含有特殊的突变,是鉴定某一特定E3下游蛋白质底物的有效手段。但E1与E2结合位点的突变设计仍有待完善。本研究重新审视了E1-E2的相互作用,着眼于泛素活化酶Ube1活性中心内的疏水区,突变了该区上3个关键的苯丙氨酸残基为丙氨酸,即F637A、F729A、F741A,并设置合理的实验对照来探究此突变对泛素从E1向E2传递活性的影响。结果表明,突变体m Ube1较好地阻断了与UbcH7的识别及Ub的传递,提示本研究涉及的3个关键苯丙氨酸位点对E1-E2互相识别的重要性,及其作为新的正交泛素传递途径中E1突变位点的合理性。     

Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.