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AlphaFold has burst into our lives. A powerful algorithm that underscores the strength of biological sequence data and artificial intelligence (AI). AlphaFold has appended projects and research directions. The database it has been creating promises an untold number of applications with vast potential impacts that are still difficult to surmise. AI approaches can revolutionize personalized treatments and usher in better-informed clinical trials. They promise to make giant leaps toward reshaping and revamping drug discovery strategies, selecting and prioritizing combinations of drug targets. Here, we briefly overview AI in structural biology, including in molecular dynamics simulations and prediction of microbiota-human protein-protein interactions. We highlight the advancements accomplished by the deep-learning-powered AlphaFold in protein structure prediction and their powerful impact on the life sciences. At the same time, AlphaFold does not resolve the decades-long protein folding challenge, nor does it identify the folding pathways. The models that AlphaFold provides do not capture conformational mechanisms like frustration and allostery, which are rooted in ensembles, and controlled by their dynamic distributions. Allostery and signaling are properties of populations. AlphaFold also does not generate ensembles of intrinsically disordered proteins and regions, instead describing them by their low structural probabilities. Since AlphaFold generates single ranked structures, rather than conformational ensembles, it cannot elucidate the mechanisms of allosteric activating driver hotspot mutations nor of allosteric drug resistance. However, by capturing key features, deep learning techniques can use the single predicted conformation as the basis for generating a diverse ensemble.

Ras GTPase interacts with its regulators and downstream effectors for its critical function in cellular signaling. Targeting the disrupted mechanisms in Ras-related human cancers requires understanding the distinct dynamics of these protein-protein interactions. We performed normal mode analysis (NMA) of KRas4B in wild-type or mutant monomeric and neurofibromin-1 (NF1), Son of Sevenless 1 (SOS1) or Raf-1 bound dimeric conformational states to reveal partner-specific dynamics of the protein. Gaussian network model (GNM) analysis showed that the known KRas4B lobes further partition into subdomains upon binding to its partners. Furthermore, KRas4B interactions with different partners suppress the flexibility in not only their binding sites but also distant residues in the allosteric lobe in a partner-specific way. The conformational changes can be driven by intrinsic residue fluctuations of the open state KRas4B-GDP, as we illustrated with anisotropic network model (ANM) analysis. The allosteric paths connecting the nucleotide binding residues to the allosteric site at alpha 3-L7 portray differences in the inactive and active states. These findings help in understanding the partner-specific KRas4B dynamics, which could be utilized for therapeutic targeting., National Cancer Institute; National Institutes of Health; Intramural Research Program of the NIH; Center for Cancer Research

Small molecules play a critical role in modulating biological systems. Knowledge of chemical-protein interactions helps address fundamental and practical questions in biology and medicine. However, with the rapid emergence of newly sequenced genes, the endogenous or surrogate ligands of a vast number of proteins remain unknown. Homology modeling and machine learning are two major methods for assigning new ligands to a protein but mostly fail when sequence homology between an unannotated protein and those with known functions or structures is low. In this study, we develop a new deep learning framework to predict chemical binding to evolutionary divergent unannotated proteins, whose ligand cannot be reliably predicted by existing methods. By incorporating evolutionary information into self-supervised learning of unlabeled protein sequences, we develop a novel method, distilled sequence alignment embedding (DISAE), for the protein sequence representation. DISAE can utilize all protein sequences and their multiple sequence alignment (MSA) to capture functional relationships between proteins without the knowledge of their structure and function. Followed by the DISAE pretraining, we devise a module-based fine-tuning strategy for the supervised learning of chemical-protein interactions. In the benchmark studies, DISAE significantly improves the generalizability of machine learning models and outperforms the state-of-the-art methods by a large margin. Comprehensive ablation studies suggest that the use of MSA, sequence distillation, and triplet pretraining critically contributes to the success of DISAE. The interpretability analysis of DISAE suggests that it learns biologically meaningful information. We further use DISAE to assign ligands to human orphan G-protein coupled receptors (GPCRs) and to cluster the human GPCRome by integrating their phylogenetic and ligand relationships. The promising results of DISAE open an avenue for exploring the chemical landscape of entire sequenced genomes.

Cobalamin riboswitch is a cis-regulatory element widely found in the 5'-UTRs of the vitamin B12-associated genes in bacteria, resulting in modulation and production of a particular protein. Thermoanaerobacter tengcongensis (Tte) AdoCbl riboswitches are the largest of the known riboswitches with 210 nucleotides, partially due to its long peripheral P6-extension, which enable high affinity of AdoCbl. Two structural elements, T-loop/T-looplike motif and kissing loop are key to the global folding of the RNA. While the structure of the TteAdoCbl riboswitch complex is known, we still do not understand the structure and conformation before AdoCbl ligand recognition. In order to delineate the conformational changes and the stabilities of long-range interactions, we have performed extensive all-atom replica-exchange molecular dynamics simulations of the TteAdoCbl riboswitch with a total simulation time of 2296 ns. We found that both the T-loop/T-looplike motif and kissing loop are very stable with ligand binding. The gating conformation changes of P6-extension allow the ligand to bind to the preorganized kissing loop binding pocket. The T-loop/T-looplike motif has much more hydrogen bonds than observed in TteAdoCbl riboswitch complex crystal structure, indicating an allosteric response of the T-loop/T-looplike motif. Our study demonstrated that the conformational ensemble of TteAdoCbl riboswitch provides stable structural elements for conformation selection and population shift in cobalamin recognition.

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca2+ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca2+-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca2+ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca2+ as a key modulator of the CaM-IQGAP1 interactions.

Rac1 is a small GTPase that plays key roles in actin reorganization, cell motility, and cell survival/growth as well as in various cancer types and neurodegenerative diseases. Similar to other Ras superfamily GTPases, Rac1 switches between active GTP-bound and inactive GDP-bound states. Switch I and II regions open and close during GDP/GTP exchange. P29S and A159V (paralogous to K-Ras(A146)) mutations are the two most common somatic mutations of Rac1. Rac1(P2)(9S)( )is a known hotspot for melanoma, whereas Rac1(A159V) most commonly occurs in head and neck cancer. To investigate how these substitutions induce the Rac1 dynamics, we used atomistic molecular dynamics simulations on the wild-type Rac1 and two mutant systems (P29S and A159V) in the GTP bound state, and on the wild-type Rac1 and P29S mutated system in the GDP bound state. Here, we show that P29S and A159V mutations activate Rac1 with different mechanisms. In Rac1(P29S)-GTP, the substitution increases the flexibility of Switch I based on RMSF and dihedral angle calculations and leads to an open conformation. We propose that the open Switch I conformation is one of the underlying reasons for rapid GDP/GTP exchange of Rac1(P29S). On the other hand, in Rac1(A159V)-GTP, some of the contacts of the guanosine ring of GTP with Rac1 are temporarily lost, enabling the guanosine ring to move toward Switch I and subsequently close the switch. Rac1(A159V)-GTP adopts a Ras state 2 like conformation, where both switch regions are in closed conformation and Thr35 forms a hydrogen bond with the nucleotide., National Cancer Institute; National Institutes of Health; Intramural Research Program of the NIH; Center for Cancer Research

PI3Kα is a key lipid kinase in the PI3K/Akt pathway. Its frequent oncogenic mutations make it a primary drug target. Calmodulin (CaM) activates PI3Kα independently of extracellular signals, indicating a significant role in oncogenic PI3Kα activation. Here, we reveal the atomic-scale structures of CaM in complexes with the nSH2 and cSH2 domains of the regulatory p85α subunit of PI3Kα, and illustrate how CaM activates PI3Kα by targeting the “soft 1–5–10” CaM-binding motifs in both nSH2 and cSH2 domains. Experiment observed CaM binding cSH2 first, followed by nSH2 binding hours later. CaM typically prefers binding helical peptides. Here we observe that, unlike in cSH2, the CaM-binding motif in nSH2 populates a mixed β-sheet/α-helix/random coil structure. The population shift from a β-sheet toward CaM’s favored α-helical conformation explains why the nSH2 domain needs a longer time for CaM binding in the experiments. The “soft” CaM-binding motifs in both nSH2 and cSH2 domains establish strong CaM–PI3Kα interactions, collectively facilitating PI3Kα activation. This work uncovers the structural basis for CaM-driven PI3Kα activation.

Self-assembly of peptides is closely related to many diseases, including Alzheimer’s, Parkinson’s, and prion diseases. Understanding the basic mechanism of this assembly is essential for designing ultimate cure and preventive measures. Template-assisted self-assembly (TASA) of peptides on inorganic substrates can provide fundamental understanding of substrate-dependent peptides assemble, including the role of hydrophobic interface on the peptide fibrillization. Here, we have studied the self-assembly process of a potential pentapeptide inhibitor on the surface of highly oriented pyrolytic graphite (HOPG) using real time atomic force microscopy (RT-AFM) as well as molecular dynamics (MD) simulation. Experimental and simulation results show nanofilament formation consisting of β-sheet structures and epitaxial growth on HOPG. Height analysis of the nanofilaments and MD simulation indicate that the peptides adopt a lying down configuration of double-layered antiparallel β-sheets for its epitaxial growth, and the number of nanofilament layers is concentration-dependent. These findings provide new perspective for the mechanism of peptide-based fibrillization in amyloid diseases as well as for designing well-ordered micrometrical and nanometrical structures.

To signal, Ras isoforms must be enriched at the plasma membrane (PM). It was suggested that phosphodiesterase-δ (PDEδ) can bind and shuttle some farnesylated Ras isoforms to the PM, but not all. Among these, interest focused on K-Ras4B, the most abundant oncogenic Ras isoform. To study PDEδ/Ras interactions, we modeled and simulated the PDEδ/K-Ras4B complex. We obtained structures, which were similar to two subsequently determined crystal structures. We next modeled and simulated complexes of PDEδ with the farnesylated hypervariable regions of K-Ras4A and N-Ras. Earlier data suggested that PDEδ extracts K-Ras4B and N-Ras from the PM, but surprisingly not K-Ras4A. Earlier analysis of the crystal structures advanced that the presence of large/charged residues adjacent to the farnesylated site precludes the PDEδ interaction. Here, we show that PDEδ can bind to farnesylated K-Ras4A and N-Ras like K-Ras4B, albeit not as strongly. This weaker binding, coupled with the stronger anchoring of K-Ras4A in the membrane (but not of electrostatically neutral N-Ras), can explain the observation why PDEδ is unable to effectively extract K-Ras4A. We thus propose that farnesylated Ras isoforms can bind PDEδ to fulfill the required PM enrichment, and argue that the different environments, PM versus solution, can resolve apparently puzzling Ras observations. These are novel insights that would not be expected based on the crystal structures alone, which provide an elegant rationale for previously puzzling observations of the differential effects of PDEδ on farnesylated Ras family proteins.

Amyloid β (Aβ) oligomers are the predominant toxic species in the pathology of Alzheimer’s disease. The prevailing mechanism for toxicity by Aβ oligomers includes ionic homeostasis destabilization in neuronal cells by forming ion channels. These channel structures have been previously studied in model lipid bilayers. In order to gain further insight into the interaction of Aβ oligomers with natural membrane compositions, we have examined the structures and conductivities of Aβ oligomers in a membrane composed of brain total lipid extract (BTLE). We utilized two complementary techniques: atomic force microscopy (AFM) and black lipid membrane (BLM) electrical recording. Our results indicate that Aβ1–42 forms ion channel structures in BTLE membranes, accompanied by a heterogeneous population of ionic current fluctuations. Notably, the observed current events generated by Aβ1–42 peptides in BTLE membranes possess different characteristics compared to current events generated by the presence of Aβ1–42 in model membranes comprised of a 1:1 mixture of DOPS and POPE lipids. Oligomers of the truncated Aβ fragment Aβ17–42 (p3) exhibited similar ion conductivity behavior as Aβ1–42 in BTLE membranes. However, the observed macroscopic ion flux across the BTLE membranes induced by Aβ1–42 pores was larger than for p3 pores. Our analysis of structure and conductance of oligomeric Aβ pores in a natural lipid membrane closely mimics the in vivo cellular environment suggesting that Aβ pores could potentially accelerate the loss of ionic homeostasis and cellular abnormalities. Hence, these pore structures may serve as a target for drug development and therapeutic strategies for AD treatment.

The pathogenesis of Parkinson’s disease is closely associated with the aggregation of the α-synuclein protein. Several familial mutants have been identified and shown to affect the aggregation kinetics of α-synuclein through distinct molecular mechanisms. Quantitative evaluation of the relative stabilities of the wild type and mutant fibrils is crucial for understanding the aggregation process and identifying the key component steps. In this work, we examined two topologically different α-synuclein fibril structures that are either determined by solid-state NMR method or modeled based on solid-state NMR data, and characterized their conformational properties and thermodynamic stabilities using molecular dynamics simulations. We show that the two fibril morphologies have comparable size, solvent exposure, secondary structures, and similar molecule/peptide binding modes; but different stabilities. Familial mutations do not significantly alter the overall fibril structures but shift their relative stabilities. Distinct mutations display altered fibril conformational behavior, suggesting different propagation preferences, reminiscent of cross-seeding among prion strains and tau deletion mutants. The simulations quantify the hydrophobic and electrostatic interactions, as well as N-terminal dynamics, that may contribute to the divergent aggregation kinetics that has been observed experimentally. Our results indicate that small molecule and peptide inhibitors may share the same binding region, providing molecular recognition that is independent of fibril conformation.

Tau proteins are hyperphosphorylated at common sites in their N- and C-terminal domains in at least three neurodegenerative diseases, Parkinson, dementia with Lewy bodies, and Alzheimer’s, suggesting specific pathology but general mechanism. Full-length human tau filament comprises a rigid core and a two-layered fuzzy coat. Tau is categorized into two groups of isoforms, with either four repeats (R1-R4) or three repeats (R1, R3, and R4); their truncated constructs are respectively called K18 and K19. Using multiscale molecular dynamics simulations, we explored the conformational consequences of hyperhposphorylation on tau’s repeats. Our lower conformational energy filament models suggest a rigid filament core with a radius of ∼30 to 40 Å and an outer layer with a thickness of ∼140 Å consisting of a double-layered polyelectrolyte. The presence of the phosphorylated terminal domains alters the relative stabilities in the K18 ensemble, thus shifting the populations of the full-length filaments. However, the structure with the straight repeats in the core region is still the most stable, similar to the truncated K18 peptide species without the N- and C-terminus. Our simulations across different scales of resolution consistently reveal that hyperphosphorylation of the two terminal domains decreases the attractive interactions among the N- and C-terminus and repeat domain. To date, the relationship on the conformational level between phosphorylation and aggregation has not been understood. Our results suggest that the exposure of the repeat domain upon hyperphosphorylation could enhance tau filament aggregation. Thus, we discovered that even though these neurodegenerative diseases vary and their associated tau filaments are phosphorylated to different extents, remarkably, the three pathologies appear to share a common tau aggregation mechanism.

Proteins are dynamic entities that undergo a plethora of conformational changes that may take place on a wide range of time scales. These changes can be as small as the rotation of one or a few side-chain dihedral angles or involve concerted motions in larger portions of the three-dimensional structure; both kinds of motions can be important for biological function and allostery. It is becoming increasingly evident that "connector regions" are important components of the dynamic personality of protein structures. These regions may be either disordered loops, i.e., poorly structured regions connecting secondary structural elements, or linkers that connect entire protein domains. Experimental and computational studies have, however, revealed that these regions are not mere connectors, and their role in allostery and conformational changes has been emerging in the last few decades. Here we provide a detailed overview of the structural properties and classification of loops and linkers, as well as a discussion of the main computational methods employed to investigate their function and dynamical properties. We also describe their importance for protein dynamics and allostery using as examples key proteins in cellular biology and human diseases such as kinases, ubiquitinating enzymes, and transcription factors.

The aggregation of the copper−zinc superoxide dismutase (SOD1) protein is linked to familial amyotrophic lateral sclerosis, a progressive neurodegenerative disease. A recent experimental study has shown that the (147)GVIGIAQ(153) SOD1 C-terminal segment not only forms amyloid fibrils in isolation but also accelerates the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks its fibril formation. Amyloid formation is a nucleation−polymerization process. In this study, we investigated the oligomerization and the nucleus structure of this heptapeptide. By performing extensive replica-exchange molecular dynamics (REMD) simulations and conventional MD simulations, we found that the GVIGIAQ hexamers can adopt highly ordered bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability and results in the disappearance of bilayer β-sheet structures and the increase of disordered hexamers. We identified mixed parallel−antiparallel bilayer β-sheets in both REMD and conventional MD simulations and provided the conformational transition from the experimentally observed parallel bilayer sheets to the mixed parallel−antiparallel bilayer β-sheets. Our simulations suggest that the critical nucleus consists of six peptide chains and two additional peptide chains strongly stabilize this critical nucleus. The stabilized octamer is able to recruit additional random peptides into the β-sheet. Therefore, our simulations provide insights into the critical nucleus formation and the smallest stable nucleus of the (147)GVIGIAQ(153) peptide.

Ras proteins are classical members of small GTPases that function as molecular switches by alternating between inactive GDP-bound and active GTP-bound states. Ras activation is regulated by guanine nucleotide exchange factors that catalyze the exchange of GDP by GTP, and inactivation is terminated by GTPase-activating proteins that accelerate the intrinsic GTP hydrolysis rate by orders of magnitude. In this review, we focus on data that have accumulated over the past few years pertaining to the conformational ensembles and the allosteric regulation of Ras proteins and their interpretation from our conformational landscape standpoint. The Ras ensemble embodies all states, including the ligand-bound conformations, the activated (or inactivated) allosteric modulated states, post-translationally modified states, mutational states, transition states, and nonfunctional states serving as a reservoir for emerging functions. The ensemble is shifted by distinct mutational events, cofactors, post-translational modifications, and different membrane compositions. A better understanding of Ras biology can contribute to therapeutic strategies.

Human HRAS, KRAS, and NRAS genes encode four isoforms of Ras, a p21 GTPase. Mutations in KRAS account for the majority of RAS-driven cancers. The KRAS has two splice variants, K-Ras4A and K-Ras4B. Due to their reversible palmitoylation, K-Ras4A and N-Ras have bimodal signaling states. K-Ras4A and K-Ras4B differ in four catalytic domain residues (G151R/D153E/K165Q/H166Y) and in their disordered C-terminal hypervariable region (HVR). In K-Ras4A, the HVR is not as strongly positively charged as in K-Ras4B (+6e vs +9e). Here, we performed all-atom molecular dynamics simulations to elucidate isoform-specific differences between the two splice variants. We observe that the catalytic domain of GDP-bound K-Ras4A has a more exposed nucleotide binding pocket than K-Ras4B, and the dynamic fluctuations in switch I and II regions also differ; both factors may influence guanine–nucleotide exchange. We further observe that like K-Kas4B, full-length K-Ras4A exhibits nucleotide-dependent HVR fluctuations; however, these fluctuations differ between the GDP-bound forms of K-Ras4A and K-Ras4B. Unlike K-Ras4B where the HVR tends to cover the effector binding region, in K-Ras4A, autoinhibited states are unstable. With lesser charge, the K-Ras4A HVR collapses on itself, making it less available for binding the catalytic domain. Since the HVRs of N- and H-Ras are weakly charged (+1e and +2e, respectively), autoinhibition may be a unique feature of K-Ras4B.

There are synergistic effects of Aβ and tau protein in Alzheimer’s disease. Aβ1–42 protofibril seeds induce conversion of human tau protein into β-sheet-rich toxic tau oligomers. However, the molecular mechanisms underlying such a conformational conversion are unclear. Here, we use extensive all atom replica exchange molecular dynamics simulations to investigate the effects of preformed Aβ1–42 protofibril on two monomeric tau constructs: K18 and K19. We found that Aβ oligomer stretches tau conformation and drastically reduces the metastable secondary structures/hydrogen bonding/salt-bridge networks in tau monomers and exposes their fibril nucleating motifs 275VQIINK280 and 306VQIVYK311. Aβ interacting patches around Tyr10/Ile41 contribute significantly to the interactions with K18 and K19. Aβ cross-seeded tau aggregation can adopt a “stretching-and-packing” mechanism, paving the way for the next, prion-like growth step. The results provide a mechanism on the atomic level to experimental observations that ...

Linking cell signaling events to the fundamental physicochemical basis of the conformational behavior of single molecules and ultimately to cellular function is a key challenge facing the life sciences. Here we outline the emerging principles of allosteric interactions in cell signaling, with emphasis on the following points. (1) Allosteric efficacy is not a function of the chemical composition of the allosteric pocket but reflects the extent of the population shift between the inactive and active states. That is, the allosteric effect is determined by the extent of preferred binding, not by the overall binding affinity. (2) Coupling between the allosteric and active sites does not decide the allosteric effect; however, it does define the propagation pathways, the allosteric binding sites, and key on-path residues. (3) Atoms of allosteric effectors can act as "driver" or "anchor" and create attractive "pulling" or repulsive "pushing" interactions. Deciphering, quantifying, and integrating the multiple co-occurring events present daunting challenges to our scientific community.

Membrane permeability to ions and small molecules is believed to be a critical step in the pathology of Alzheimer’s disease (AD). Interactions of oligomers formed by amyloid-β (Aβ) peptides with the plasma cell membrane are believed to play a fundamental role in the processes leading to membrane permeability. Among the family of Aβs, pyroglutamate (pE)-modified Aβ peptides constitute the most abundant oligomeric species in the brains of AD patients. Although membrane permeability mechanisms have been studied for full-length Aβ1–40/42 peptides, these have not been sufficiently characterized for the more abundant AβpE3–42 fragment. Here we have compared the adsorbed and membrane-inserted oligomeric species of AβpE3–42 and Aβ1–42 peptides. We find lower concentrations and larger dimensions for both species of membrane-associated AβpE3–42 oligomers. The larger dimensions are attributed to the faster self-assembly kinetics of AβpE3–42, and the lower concentrations are attributed to weaker interactions with zwitterionic lipid headgroups. While adsorbed oligomers produced little or no significant membrane structural damage, increased membrane permeabilization to ionic species is understood in terms of enlarged membrane-inserted oligomers. Membrane-inserted AβpE3–42 oligomers were also found to modify the mechanical properties of the membrane. Taken together, our results suggest that membrane-inserted oligomers are the primary species responsible for membrane permeability.

Among the family of Aβ peptides, pyroglutamate-modified Aβ (AβpE) peptides are particularly associated with cytotoxicity in Alzheimer's disease (AD). They represent the dominant fraction of Aβ oligomers in the brains of AD patients, but their accumulation in the brains of elderly individuals with normal cognition is significantly lower. Accumulation of AβpE plaques precedes the formation of plaques of full-length Aβ (Aβ1-40/42). Most of these properties appear to be associated with the higher hydrophobicity of AβpE as well as an increased resistance to enzymatic degradation. However, the important question of whether AβpE peptides induce pore activity in lipid membranes and their potential toxicity compared with other Aβ pores is still open. Here we examine the activity of AβpE pores in anionic membranes using planar bilayer electrical recording and provide their structures using molecular dynamics simulations. We find that AβpE pores spontaneously induce ionic current across the membrane and have some similar properties to the other previously studied pores of the Aβ family. However, there are also some significant differences. The onset of AβpE3-42 pore activity is generally delayed compared with Aβ1-42 pores. However, once formed, AβpE3-42 pores produce increased ion permeability of the membrane, as indicated by a greater occurrence of higher conductance electrical events. Structurally, the lactam ring of AβpE peptides induces a change in the conformation of the N-terminal strands of the AβpE3-42 pores. While the N-termini of wild-type Aβ1-42 peptides normally reside in the bulk water region, the N-termini of AβpE3-42 peptides tend to reside in the hydrophobic lipid core. These studies provide a first step to an understanding of the enhanced toxicity attributed to AβpE peptides.

An unusual ΔE693 mutation in the amyloid precursor protein (APP) producing a β-amyloid (Aβ) peptide lacking glutamic acid at position 22 (Glu22) was recently discovered, and dabbed the Osaka mutant (ΔE22). Previously, several point mutations in the Aβ peptide involving Glu22 substitutions were identified and implicated in the early onset of familial Alzheimer’s disease (FAD). Despite the absence of Glu22, the Osaka mutant is also associated with FAD, showing a recessive inheritance in families affected by the disease. To see whether this aggregation-prone Aβ mutant could directly relate to the Aβ ion channel-mediated mechanism as observed for the wild type (WT) Aβ peptide in AD pathology, we modeled Osaka mutant β-barrels in a lipid bilayer. Using molecular dynamics (MD) simulations, two conformer ΔE22 barrels with the U-shaped monomer conformation derived from NMR-based WT Aβ fibrils were simulated in explicit lipid environment. Here, we show that the ΔE22 barrels obtain the lipid-relaxed β-sheet channel topology, indistinguishable from the WT Aβ1–42 barrels, as do the outer and pore dimensions of octadecameric (18-mer) ΔE22 barrels. Although the ΔE22 barrels lose the cationic binding site in the pore which is normally provided by the negatively charged Glu22 side-chains, the mutant pores gain a new cationic binding site by Glu11 at the lower bilayer leaflet, and exhibit ion fluctuations similar to the WT barrels. Of particular interest, this deletion mutant suggests that toxic WT Aβ1–42 would preferentially adopt a less C-terminal turn similar to that observed for Aβ17–42, and explains why the solid state NMR data for Aβ1–40 point to a more C-terminal turn conformation. The observed ΔE22 barrels conformational preferences also suggest an explanation for the lower neurotoxicity in rat primary neurons as compared to WT Aβ1–42.

Cellular functions are performed through protein-protein interactions; therefore, identification of these interactions is crucial for understanding biological processes. Recent studies suggest that knowledge-based approaches are more useful than "blind" docking for modeling at large scales. However, a caveat of knowledge-based approaches is that they treat molecules as rigid structures. The Protein Data Bank (PDB) offers a wealth of conformations. Here, we exploited an ensemble of the conformations in predictions by a knowledge-based method, PRISM. We tested "difficult" cases in a docking-benchmark data set, where the unbound and bound protein forms are structurally different. Considering alternative conformations for each protein, the percentage of successfully predicted interactions increased from ~26 to 66%, and 57% of the interactions were successfully predicted in an "unbiased" scenario, in which data related to the bound forms were not utilized. If the appropriate conformation, or relevant template interface, is unavailable in the PDB, PRISM could not predict the interaction successfully. The pace of the growth of the PDB promises a rapid increase of ensemble conformations emphasizing the merit of such knowledge-based ensemble strategies for higher success rates in protein-protein interaction predictions on an interactome scale. We constructed the structural network of ERK interacting proteins as a case study.

Tau pathology in Alzheimer’s disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron–electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand–turn−β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles.

Alzheimer's disease (AD) is the most common type of senile dementia in aging populations. Amyloid β (Aβ)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aβ-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aβ binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aβ peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aβ effects even in the absence of receptor-peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aβ isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aβ isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aβ isomer channels, and the d-isomer channel conductance is blocked by Zn(2+), a known blocker of l-Aβ isomer channels. MD simulations further verify formation of β-barrel-like Aβ channels with d- and l-isomers, illustrating that both d- and l-Aβ barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca(2+) leaking, unregulated channels in AD, and suggest that Aβ toxicity is mediated through a receptor-independent, nonstereoselective mechanism.

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes.

Ubiquitination is crucial for many cellular processes such as protein degradation, DNA repair, transcription regulation, and cell signaling. Ubiquitin attachment takes place via a sequential enzymatic cascade involving ubiquitin activation (by El enzymes), ubiquitin conjugation (by E2 enzymes), and ubiquitin substrate tagging (by E3 enzymes). E3 ligases mediate ubiquitin transfer from E2s to substrates and as such confer substrate specificity. Although E3s can interact and function with numerous E2s, it is still unclear how they choose which E2 to use. Identifying all E2 partners of an E3 is essential for inferring the principles guiding E2 selection by an E3. Here we model the interactions of E3 and E2 proteins in a large, proteome-scale strategy based on interface structural motifs, which allows elucidation of (1) which E3s interact with which E2s in the human ubiquitination pathway and (2) how they interact with each other. Interface analysis of E2-E3 complexes reveals that loop L1 of E2s is critical for binding; the residue in the sixth position in loop L1 is widely utilized as an interface hot spot and appears indispensible for E2 interactions. Other loop L1 residues also confer specificity on the E2-E3 interactions: HECT E3s are in contact with the residue in the second position in loop L1 of E2s, but this is not the case for the RING finger type E3s. Our modeled E2-E3 complexes illuminate how slight sequence variations in E2 residues may contribute to specificity in E3 binding. These findings may be important for discovering drug candidates targeting E3s, which have been implicated in many diseases.

More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape β-strand-turn-β-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids are not only toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.

Photopolymerizable phospholipid DC(8,9)PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Because of the presence of the diacetylene groups, DC(8,9)PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC(8,9)PC photopolymerization in gel-phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-vis absorption spectroscopy occurred within 2 min after UV treatment, whereas no spectral shifts were observed when DC(8,9)PC was incorporated into liquid-phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in the amount of DC(8,9)PC monomer in both DPPC and POPC environments without any change in the matrix lipids in UV-treated samples. Molecular dynamics (MD) simulations of DPPC/DC(8,9)PC and POPC/DC(8,9)PC bilayers indicate that the DC(8,9)PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, the motions of DC(8,9)PC in the gel-phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC(8,9)PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization because of the lack of appropriate alignment of the DC(8,9)PC fatty acyl chains. Fluorescence microscopy data indicates the homogeneous distribution of lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC(8,9)PC monolayers but domain formation in DPPC/DC(8,9)PC monolayers. These results show that the DC(8,9)PC molecules cluster and assume the preferred conformation in the gel-phase matrix for the UV-triggered polymerization reaction.

Amyloid plaques and neurofibrillary tangles simultaneously accumulate in Alzheimer's disease (AD). It is known that Aβ and tau exist together in the mitochondria; however, the interactions between Aβ oligomers and tau are controversial. Moreover, it is still unclear which specific domains in the tau protein can interact with Aβ oligomers and what could be the effect of these interactions. Herein, we examine three different Aβ-tau oligomeric complexes. These complexes present interactions of Aβ with three domains in the tau protein; all contain high β-structure propensity in their R2, R3, and R4 repeats. Our results show that, among these, Aβ oligomers are likely to interact with the R2 domain to form a stable complex with better alignment in the turn region and the β-structure domain. We therefore propose that the R2 domain can interact with soluble Aβ oligomers and consequently promote aggregation. EM and AFM images and dimensions revealed highly polymorphic tau aggregates. We suggest that the polymorphic tau and Aβ-tau aggregates may be largely due to repeat sequences which are prone to variable turn locations along the tau repeats.

Cu(2+) binding to Alzheimer's β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.

Elucidating the structure of Aβ(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Aβ(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across β-strands and across β-sheets in Aβ fibrils. Experimentally, data for the three-fold symmetry structure of the Aβ(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-β units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Aβ(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed ∼5-7 Å M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 Å. Consequently, we revealed that the unique Aβ(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Aβ(1-42) fibrils, point to the relevance of a cavity in Aβ(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity.

Glycogen synthase kinase 3β (GSK-3β) is a serine-threonine kinase belonging to the CMGC family that plays a key role in many biological processes, such as glucose metabolism, cell cycle regulation, and proliferation. Like most protein kinases, GSK-3β is regulated via multiple pathways and sites. We performed all-atom molecular dynamics simulations on the unphosphorylated and phosphorylated unbound GSK-3β and the phosphorylated GSK-3β bound to a peptide substrate, its product, and a derived inhibitor. We found that GSK-3β autophosphorylation at residue Tyr(216) results in widening of the catalytic groove, thereby facilitating substrate access. In addition, we studied the interactions of the phosphorylated GSK-3β with a substrate and peptide inhibitor located at the active site and observed higher affinity of the inhibitor to the kinase. Furthermore, we detected a potential remote binding site which was previously identified in other kinases. In agreement with experiments we observed that binding of specific peptides at this remote site leads to stabilization of the activation loop located in the active site. We speculate that this stabilization could enhance the catalytic activity of the kinase. We point to this remote site as being structurally conserved and suggest that the allosteric phenomenon observed here may occur in the protein kinase superfamily.

Water molecules play a major role in the P450 catalytic cycle. Here, we locate the preferred water pathways and their gating mechanisms for the human cytochrome P450 3A4 (CYP3A4) and elucidate the role of the cytochrome P450 reductase (CPR) in turning on and activating these water channels. We perform explicit solvent molecular dynamic simulations of CYP3A4, unbound and bound to two substrates, and with and without the flavin mononucleotide (FMN)-binding domain of CPR. We observe in/out passage of water molecules via a water-specific and conserved channel (aqueduct) located between the active site and the heme proximal side. We find that the aqueduct gating mechanism is mediated by R375, the conserved arginine that salt bridges with the heme 7-propionate. When R375 rotates, it opens the aqueduct and establishes a connection between a cluster of active site water molecules network and the bulk solvent. The aqueduct region overlaps with the CPR binding-site to CYP3A4. Indeed, we find that when the FMN domain of CPR binds to CYP3A4, the aqueduct fully opens up, thereby allowing a flow of water molecules. The aqueduct's opening can permit proton transfer, shuttling the protons to the active site through ordered water molecules. In addition, the expulsion of water molecules via the aqueduct contributes to substrate binding. As such, the CPR binding has a function: it triggers the aqueduct's opening and thereby enables a proton shuttle pathway, which is needed for the dioxygen activation. This mechanism could be a general paradigm in P450s.

Virtual screening is emerging as a productive and cost-effective technology in rational drug design for the identification of novel lead compounds. An important model for virtual screening is the pharmacophore. Pharmacophore is the spatial configuration of essential features that enable a ligand molecule to interact with a specific target receptor. In the absence of a known receptor structure, a pharmacophore can be identified from a set of ligands that have been observed to interact with the target receptor. Here, we present a novel computational method for pharmacophore detection and virtual screening. The pharmacophore detection module is able to (i) align multiple flexible ligands in a deterministic manner without exhaustive enumeration of the conformational space, (ii) detect subsets of input ligands that may bind to different binding sites or have different binding modes, (iii) address cases where the input ligands have different affinities by defining weighted pharmacophores based on the number of ligands that share them, and (iv) automatically select the most appropriate pharmacophore candidates for virtual screening. The algorithm is highly efficient, allowing a fast exploration of the chemical space by virtual screening of huge compound databases. The performance of PharmaGist was successfully evaluated on a commonly used data set of G-Protein Coupled Receptor alpha1A. Additionally, a large-scale evaluation using the DUD (directory of useful decoys) data set was performed. DUD contains 2950 active ligands for 40 different receptors, with 36 decoy compounds for each active ligand. PharmaGist enrichment rates are comparable with other state-of-the-art tools for virtual screening.

Beta(2)-microglobulin (beta(2)m) amyloid deposits are linked to dialysis-related amyloidosis (DRA) in hemodialysis patients. The mechanism by which beta(2)m causes DRA is not understood. It is also unclear whether only the full-length beta(2)m induces pathophysiology or if proteolytic fragments are sufficient for inducing this effect. Ser20-Lys41 (K3) is a digestion fragment of full-length beta(2)m. Solid state NMR (ssNMR) combined with X-ray diffraction and atomic force microscopy (AFM) revealed the characteristic oligomeric amyloid conformation of the U-turn beta-strand-turn-beta-strand motif stacked in parallel and stabilized by intermolecular interactions also shown by Abeta(9-40)/Abeta(17-42) and the CA150 WW domain. Here we use the K3 U-turn atomic coordinates and molecular dynamic (MD) simulations to model K3 channels in the membrane. Consistent with previous AFM imaging of other amyloids that show channel-like structures in the membrane, in the simulations K3 also forms ion channels with 3-6 loosely attached mobile subunits. We carry out AFM, single channel electrical recording, and fluorescence imaging experiments. AFM images display 3D ion channel topography with shapes, morphologies, and dimensions consistent with the theoretical model. Electrical conductance measurements indicate multiple single channel conductances, suggesting that various K3 oligomer sizes can constitute the channel structure. Fluorescence measurements in kidney cells show channel-mediated cell calcium uptake. These results suggest that the beta(2)m-induced DRA can be mediated by ion channels formed by its K3 fragment. Because the beta-strand-turn-beta-strand motif appears to be a universal amyloid feature, its ability to form ion channels further suggests that the motif may play a generic role in toxicity.

The human cytochrome P450 3A4 mono-oxygenates approximately 50% of all drugs. Its substrates/products enter/leave the active site by access/exit channels. Here, we perform steered molecular dynamics simulations, pulling the products temazepam and testosterone-6betaOH out of the P450 3A4 enzyme in order to identify the preferred substrate/product pathways and their gating mechanism. We locate six different egress pathways of products from the active site with different exit preferences for the two products and find that there is more than just one access/exit channel in CYP3A4. The so-called solvent channel manifests the largest opening for both tested products, thereby identifying this channel as a putative substrate channel. Most channels consist of one or two pi-stacked phenylalanine residues that serve as gate keepers. The oxidized drug breaks the hydrophobic interactions of the gating residues and forms mainly hydrophobic contacts with the gate. We argue that product exit preferences in P450s are regulated by protein-substrate specificity.

A new amino acid has been designed as a replacement for arginine (Arg, R) to protect the tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) from proteases. This amino acid, denoted (Pro)hArg, is characterized by a proline skeleton bearing a specifically oriented guanidinium side chain. This residue combines the ability of Pro to induce turn-like conformations with the Arg side-chain functionality. The conformational profile of the CREKA analogue incorporating this Arg substitute has been investigated by a combination of simulated annealing and molecular dynamics. Comparison of the results with those previously obtained for the natural CREKA shows that (Pro)hArg significantly reduces the conformational flexibility of the peptide. Although some changes are observed in the backbone...backbone and side-chain...side-chain interactions, the modified peptide exhibits a strong tendency to accommodate turn conformations centered at the (Pro)hArg residue and the overall shape of the molecule in the lowest energy conformations characterized for the natural and the modified peptides exhibit a high degree of similarity. In particular, the turn orients the backbone such that the Arg, Glu, and Lys side chains face the same side of the molecule, which is considered important for bioactivity. These results suggest that replacement of Arg by (Pro)hArg in CREKA may be useful in providing resistance against proteolytic enzymes while retaining conformational features which are essential for tumor-homing activity.

The intrinsic conformational preferences of C (alpha,alpha)-dibenzylglycine, a symmetric alpha,alpha-dialkylated amino acid bearing two benzyl substituents on the alpha-carbon atom, have been determined using quantum chemical calculations at the B3LYP/6-31+G(d,p) level. A total of 46 minimum energy conformations were found for the N-acetyl- N'-methylamide derivative, even though only nine of them showed a relative energy lower than 5.0 kcal/mol. The latter involves C 7, C 5, and alpha' backbone conformations stabilized by intramolecular hydrogen bonds and/or N-H...pi interactions. Calculation of the conformational free energies in different environments (gas-phase, carbon tetrachloride, chloroform, methanol, and water solutions) indicates that four different minima (two C 5 and two C 7) are energetically accessible at room temperature in the gas phase, while in methanol and aqueous solutions one such minimum (C 5) becomes the only significant conformation. Comparison with results recently reported for C (alpha,alpha)-diphenylglycine indicates that substitution of phenyl side groups by benzyl enhances the conformational flexibility leading to (i) a reduction of the strain of the peptide backbone and (ii) alleviating the repulsive interactions between the pi electron density of the phenyl groups and the lone pairs of the carbonyl oxygen atoms.

Specific p53 binding-induced DNA bending and its underlying responsible forces are crucial for the understanding of selective transcription activation. Diverse p53-response elements exist in the genome; however, it is not known what determines the DNA bending and to what extent. In order to gain knowledge of the forces that govern the DNA bending, molecular dynamics simulations were performed on a series of p53 core domain tetramer-DNA complexes in which each p53 core domain was bound to a DNA quarter site specifically. By varying the sequence of the central 4-base pairs of each half-site, different DNA bending extents were observed. The analysis showed that the dimer-dimer interactions in p53 were similar for the complexes; on the other hand, the specific interactions between the p53 and DNA, including the interactions of Arg280, Lys120, and Arg248 with the DNA, varied more significantly. In particular, the Arg280 interactions were better maintained in the complex with the CATG-containing DNA sequence and were mostly lost in the complex with the CTAG-containing DNA sequence. Structural analysis shows that the base pairings for the CATG sequence were stable throughout the simulation trajectory, whereas those for the CTAG sequence were partially dissociated in part of the trajectory, which affected the stability of the nearby Arg280-Gua base interactions. Thus, DNA bending depends on the balance between the p53 dimer-dimer interactions and p53-DNA interactions, which is in turn related to the DNA sequence and DNA flexibility.

We report all-atom molecular dynamics simulations of annular beta-amyloid (17-42) structures, single- and double-layered, in solution. We assess the structural stability and association force of Abeta annular oligomers associated through different interfaces, with a mutated sequence (M35A), and with the oxidation state (M35O). Simulation results show that single-layered annular models display inherent structural instability: one is broken down into linear-like oligomers, and the other collapses. On the other hand, a double-layered annular structure where the two layers interact through their C-termini to form an NC-CN interface (where N and C are the N and C termini, respectively) exhibits high structural stability over the simulation time due to strong hydrophobic interactions and geometrical constraints induced by the closed circular shape. The observed dimensions and molecular weight of the oligomers from atomic force microscopy (AFM) experiments are found to correspond well to our stable double-layered model with the NC-CN interface. Comparison with K3 annular structures derived from the beta 2-microglobulin suggests that the driving force for amyloid formation is sequence specific, strongly dependent on side-chain packing arrangements, structural morphologies, sequence composition, and residue positions. Combined with our previous simulations of linear-like Abeta, K3 peptide, and sup35-derived GNNQQNY peptide, the annular structures provide useful insight into oligomeric structures and driving forces that are critical in amyloid fibril formation.

DFT calculations at the B3LYP/6-31+G(d,p) level have been used to investigate how the replacement of the alpha hydrogen by a more sterically demanding group affects the conformational preferences of proline. Specifically, the N-acetyl-N'-methylamide derivatives of L-proline, L-alpha-methylproline, and L-alpha-phenylproline have been calculated, with both the cis/trans isomerism of the peptide bonds and the puckering of the pyrrolidine ring being considered. The effects of solvation have been evaluated by using a Self-Consistent Reaction Field model. As expected, tetrasubstitution at the alpha carbon destabilizes the conformers with one or more peptide bonds arranged in cis. The lowest energy minimum has been found to be identical for the three compounds investigated, but important differences are observed regarding other energetically accessible backbone conformations. The results obtained provide evidence that the distinct steric requirements of the substituent at C (alpha) may play a significant role in modulating the conformational preferences of proline.

Conformationally restricted amino acids are promising candidates to serve as basic pieces in redesigned protein motifs which constitute the basic modules in synthetic nanoconstructs. Here we study the ability of constrained cyclic amino acid 1-aminocyclohexane-1-carboxylic acid (Ac6c) to stabilize highly regular beta-helical motifs excised from naturally occurring proteins. Calculations indicate that the conformational flexibility observed in both the ring and the main chain is significantly higher than that detected for other 1-aminocycloalkane-1-carboxylic acids (Acnc, where n refers to the size of the ring) with smaller cycles. Incorporation of Ac6c into the flexible loops of beta-helical motifs indicates that the stability of such excised building blocks as well as the nanoassemblies derived from them is significantly enhanced. Thus, the intrinsic Ac6c tendency to adopt folded conformations combined with the low structural strain of the cyclohexane ring confers the ability to both self-adapt to the beta-helix motif and to stabilize the overall structure by absorbing part of its conformational fluctuations. Comparison with other Acnc residues indicates that the ability to adapt to the targeted position improves considerably with the ring size, i.e., when the rigidity introduced by the strain of the ring decreases.

DFT calculations at the B3LYP/6-311G(d,p) level have been used to investigate the intrinsic conformational preferences of 1-amino-2-phenylcyclopentane-1-carboxylic acid (c5Phe), a constrained analogue of phenylalanine in which the alpha and beta carbons are included in a cyclopentane ring. Specifically, the N-acetyl-N'-methylamide derivatives of the cis and trans stereoisomers, where cis and trans refer to the relative position between the amino group and the phenyl ring, have been calculated. Solvent effects have been examined using a self-consistent reaction field (SCRF) method. Results indicate that the conformational space of the cis stereoisomer is much more restricted than that of the trans derivative both in the gas phase and in solution.

Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron-oxo species, called Compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substrate-free or containing one and two molecules of diazepam as a substrate. Mössbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe-S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH---S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe-S bond, and as such, the presence of the substrate leads to a shorter Fe-S bond and it prevents the localization of the radical on the sulfur. This diazepam-Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (called also cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues.

In this work we used atomistic molecular dynamics simulations to examine different aspects of tubular nanostructures constructed using protein building blocks with a beta-helical conformation. Initially, we considered two different natural protein building blocks, which were extracted from the protein data base, to compare the relative stabilities of the nanotubes obtained made of self-assembled and covalently linked repeats. Results show nanotubes constructed by linking building blocks through covalent bonds are very stable suggesting that the basic principles of polymer physics are valid when the repeating units are made of large fragments of proteins. In contrast, the stability of self-assembled nanostructures strongly depends on the attractive nonbonding interactions associated to building blocks aligned in a complementary manner. On the other hand, we investigated the ability of a conformationally constrained synthetic amino acid to enhance the stability of both self-assembled and polymerized nanotubes when it is used to substitute natural residues. Specifically, we considered 1-aminocyclopentane-1-caboxylic acid, which involves strong stereochemical constraints produced by the cyclopentane side chain. We found that the incorporation of this amino acid within the more flexible regions of the beta-helical building blocks is an excellent strategy to enhance the stability of the nanotubes. Thus, when a single mutation is performed in the loop region of the beta-helix, the bend architecture of the whole loop is stabilized since the conformational mobility is reduced not only at the mutated position but also at the adjacent positions.

7 pages, 4 figures, 1 table., A highly constrained amino acid has been introduced in the turn region of a β-helix to increase the conformational stability of the native fold for nanotechnological purposes. The influence of this specific amino acid replacement in the final organization of β-helix motifs has been evaluated by combining ab initio first-principles calculations on model systems and molecular dynamics simulations of entire peptide segments. The former methodology, which has been applied to a sequence containing three amino acids, has been used to develop adjusted templates. Calculations indicated that 1-amino-2,2-diphenylcyclopropanecarboxylic acid, a constrained cyclopropane analogue of phenylalanine, exhibits a strong tendency to form and promote folded conformations. On the other hand, molecular dynamics simulations are employed to probe the ability of such a synthetic amino acid to enhance the conformational stability of the β-helix motif, which is the first requirement for further protein nanoengineering. A highly regular segment from a naturally occurring β-helix protein was selected as a potential nanoconstruct module. Simulations of wild type and mutated segments revealed that the ability of the phenylalanine analogue to nucleate turn conformations enhances the conformational stability of the β-helix motif in isolated peptide segments., We acknowledge the National Cancer Institute for partial allocation of computing time and staff support at the Advanced Biomedical Computing Center of the Frederick Cancer Research and Development Center. D.Z. thanks the Ramon y Cajal program of the Spanish MEC for financial support. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO- 12400. This research was supported [in part] by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.