Development of unified statistical potentials describing protein-protein interactions

A residue-based and a heavy atom-based statistical pair potential are developed for use in assessing the strength of protein-protein interactions. To ensure the quality of the potentials, a nonredundant, high-quality dimer database is constructed. The protein complexes in this dataset are checked by a literature search to confirm that they form multimers, and the pairwise amino acid preference to interact across a protein-protein interface is analyzed and pair potentials constructed. The performance of the residue-based potentials is evaluated by using four jackknife tests and by assessing the potentials' ability to select true protein-protein interfaces from false ones. Compared to potentials developed for monomeric protein structure prediction, the interdomain potential performs much better at distinguishing protein-protein interactions. The potential developed from homodimer interfaces is almost the same as that developed from heterodimer interfaces with a correlation coefficient of 0.92. The residue-based potential is well suited for genomic scale protein interaction prediction and analysis, such as in a recently developed threading-based algorithm, MULTIPROSPECTOR. However, the more time-consuming atom-based potential performs better in identifying near-native structures from docking generated decoys.     

A distance-dependent atomic knowledge-based potential for improved protein structure selection.

A heavy atom distance-dependent knowledge-based pairwise potential has been developed. This statistical potential is first evaluated and optimized with the native structure z-scores from gapless threading. The potential is then used to recognize the native and near-native structures from both published decoy test sets, as well as decoys obtained from our group's protein structure prediction program. In the gapless threading test, there is an average z-score improvement of 4 units in the optimized atomic potential over the residue-based quasichemical potential. Examination of the z-scores for individual pairwise distance shells indicates that the specificity for the native protein structure is greatest at pairwise distances of 3.5-6.5 A, i.e., in the first solvation shell. On applying the current atomic potential to test sets obtained from the web, composed of native protein and decoy structures, the current generation of the potential performs better than residue-based potentials as well as the other published atomic potentials in the task of selecting native and near-native structures. This newly developed potential is also applied to structures of varying quality generated by our group's protein structure prediction program. The current atomic potential tends to pick lower RMSD structures than do residue-based contact potentials. In particular, this atomic pairwise interaction potential has better selectivity especially for near-native structures. As such, it can be used to select near-native folds generated by structure prediction algorithms as well as for protein structure refinement.     

Brilliant blue R dye is capable of suppressing amyloid fibril formation of lysozyme

Amyloid fibril formation is associated with an array of degenerative diseases. While no real cure is currently available, evidence suggests that suppression of amyloid fibrillogenesis is an effective strategy toward combating these diseases. Brilliant blue R (BBR), a disulfonated triphenylmethane compound, has been shown to interact with fibril-forming proteins but exert different effects on amyloid fibrillogenesis. These inconsistent findings prompted us to further evaluate BBR’s effect on the inhibition/suppresion of protein fibrillogenesis. Using 129-residue hen lysozyme, which shares high sequence homology to human lysozyme associated with hereditary non-neuropathic systemic amyloidosis, as a model, this study is aimed at thoroughly examining the influence of BBR on the in vitro protein fibrillogenesis. We first showed that BBR dose-dependently attenuated lysozyme fibril formation probably by affecting the fibril growth rate, with the value of IC₅₀ determined to be ~4.39 μM. Next, we employed tryptophan fluorescence quenching method to determine the binding constant and number of binding site(s) associated with BBR-lysozyme binding. In addition, we further conducted molecular docking studies to gain a better understanding of the possible binding site(s) and interaction(s) between lysozyme and BBR. We believe some of the information and/or knowledge concerning the structure–function relationship associated with BBR’s suppressing activity obtained here can be applied for the future work in the subject matter related with the therapeutic strategies for amyloid diseases.     

Effects of amino acid composition, finite size of proteins, and sparse statistics on distance-dependent statistical pair potentials

Statistical distance dependent pair potentials are frequently used in a variety of folding, threading, and modeling studies of proteins. The applicability of these types of potentials is tightly connected to the reliability of statistical observations. We explored the possible origin and extent of false positive signals in statistical potentials by analyzing their distance dependence in a variety of randomized protein-like models. While on average potentials derived from such models are expected to equal zero at any distance, we demonstrate that systematic and significant distortions exist. These distortions originate from the limited statistical counts in local environments of proteins and from the limited size of protein structures at large distances. We suggest that these systematic errors in statistical potentials are connected to the dependence of amino acid composition on protein size and to variation in protein sizes. Additionally, atom-based potentials are dominated by a false positive signal that is due to correlation among distances measured from atoms of one residue to atoms of another residue. The significance of residue-based pairwise potentials at various spatial pair separations was assessed in this study and it was found that as few as approximately 50% of potential values were statistically significant at distances below 4 A, and only at most approximately 80% of them were significant at larger pair separations. A new definition for reference state, free of the observed systematic errors, is suggested. It has been demonstrated to generate statistical potentials that compare favorably to other publicly available ones.     

Integrating atom-based and residue-based scoring functions for protein-protein docking

Most scoring functions for protein-protein docking algorithms are either atom-based or residue-based, with the former being able to produce higher quality structures and latter more tolerant to conformational changes upon binding. Earlier, we developed the ZRANK algorithm for reranking docking predictions, with a scoring function that contained only atom-based terms. Here we combine ZRANK's atom-based potentials with five residue-based potentials published by other labs, as well as an atom-based potential IFACE that we published after ZRANK. We simultaneously optimized the weights for selected combinations of terms in the scoring function, using decoys generated with the protein-protein docking algorithm ZDOCK. We performed rigorous cross validation of the combinations using 96 test cases from a docking benchmark. Judged by the integrative success rate of making 1000 predictions per complex, addition of IFACE and the best residue-based pair potential reduced the number of cases without a correct prediction by 38 and 27% relative to ZDOCK and ZRANK, respectively. Thus combination of residue-based and atom-based potentials into a scoring function can improve performance for protein-protein docking. The resulting scoring function is called IRAD (integration of residue- and atom-based potentials for docking) and is available at http://zlab.umassmed.edu.     

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.     

Cooperative hydrogen bonding in amyloid formation

Amyloid diseases, including Alzheimer's and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.     

Localized amyloids important in diseases outside the brain--lessons from the islets of Langerhans and the thoracic aorta

It has long been understood that amyloids can be lethal in systemic diseases. More recently, it has been accepted that local cerebral aggregation of the small peptide Aβ is involved in the pathogenesis of Alzheimer's disease. Protein aggregation, with the generation of small amyloid deposits in specific organs, also occurs outside the central nervous system and often is associated with increased cell death. In this review, we discuss two lesser known but common localized amyloid fibril-forming proteins: the polypeptide hormone islet amyloid polypeptide (IAPP) and the lactadherin-derived peptide medin. IAPP aggregates and induces the depletion of islet β-cells in type 2 diabetes and in islets transplanted into type 1 diabetic subjects. Initial amyloid deposition occurs intracellularly and parts of this amyloid consist of proIAPP. Medin derived from lactadherin expressed by smooth muscle cells aggregates into amyloid in certain arteries, particularly the thoracic aortic media layer, and may have a role in the generation of the potentially lethal conditions of thoracic aortic aneurysm and dissection.     

MetAmyl: A METa-Predictor for AMYLoid Proteins

The aggregation of proteins or peptides in amyloid fibrils is associated with a number of clinical disorders, including Alzheimer''s, Huntington''s and prion diseases, medullary thyroid cancer, renal and cardiac amyloidosis. Despite extensive studies, the molecular mechanisms underlying the initiation of fibril formation remain largely unknown. Several lines of evidence revealed that short amino-acid segments (hot spots), located in amyloid precursor proteins act as seeds for fibril elongation. Therefore, hot spots are potential targets for diagnostic/therapeutic applications, and a current challenge in bioinformatics is the development of methods to accurately predict hot spots from protein sequences. In this paper, we combined existing methods into a meta-predictor for hot spots prediction, called MetAmyl for METapredictor for AMYLoid proteins. MetAmyl is based on a logistic regression model that aims at weighting predictions from a set of popular algorithms, statistically selected as being the most informative and complementary predictors. We evaluated the performances of MetAmyl through a large scale comparative study based on three independent datasets and thus demonstrated its ability to differentiate between amyloidogenic and non-amyloidogenic polypeptides. Compared to 9 other methods, MetAmyl provides significant improvement in prediction on studied datasets. We further show that MetAmyl is efficient to highlight the effect of point mutations involved in human amyloidosis, so we suggest this program should be a useful complementary tool for the diagnosis of these diseases.     

Discordant and chameleon sequences: their distribution and implications for amyloidogenicity

Identification of ambiguous encoding in protein secondary structure is paramount to develop an understanding of key protein segments underlying amyloid diseases. We investigate two types of structurally ambivalent peptides, which were hypothesized in the literature as indicators of amyloidogenic proteins: discordant α-helices and chameleon sequences. Chameleon sequences are peptides discovered experimentally in different secondary-structure types. Discordant α-helices are α-helical stretches with strong β-strand propensity or prediction. To assess the distribution of these features in known protein structures, and their potential role in amyloidogenesis, we analyzed the occurrence of discordant α-helices and chameleon sequences in nonredundant sets of protein domains (n = 4263) and amyloidogenic proteins extracted from the literature (n = 77). Discordant α-helices were identified if discordance was observed between known secondary structures and secondary-structure predictions from the GOR-IV and PSIPRED algorithms. Chameleon sequences were extracted by searching for identical sequence words in α-helices and β-strands. We defined frustrated chameleons and very frustrated chameleons based on varying degrees of total β propensity ≥α propensity. To our knowledge, this is the first study to discern statistical relationships between discordance, chameleons, and amyloidogenicity. We observed varying enrichment levels for some categories of discordant and chameleon sequences in amyloidogenic sequences. Chameleon sequences are also significantly enriched in proteins that have discordant helices, indicating a clear link between both phenomena. We identified the first set of discordant-chameleonic protein segments we predict may be involved in amyloidosis. We present a detailed analysis of discordant and chameleons segments in the family of one of the amyloidogenic proteins, the Prion Protein.     

Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function?

Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow, off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation.     

Conformational preferences of non-polar amino acid residues: an additional factor in amyloid formation

Amyloid consists of β-sheet polymers and is associated with disease and with functional assemblies. Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation. The most non-polar residues promote either β-strands (Val, Ile, Phe, and Cys, VIFC) or α-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only α-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known. Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM. For the amyloid β-peptide associated with Alzheimer’s disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of β-sheet aggregates and amyloid, while LAM bias prevents it. VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions.     

Preventing protein aggregation by its hyper-acidic fusion cognates in Escherichia coli

Preventing protein aggregation is crucial for various protein studies, and has a large potential for remedy of protein misfolding or aggregates-linked diseases. In this study, we demonstrated the hyper-acidic protein fusion partners, which were previously reported to enhance the soluble expression of aggregation-prone proteins, could also significantly prevent aggregation (or improve the solubility) of disease-associated and amyloid/fibril-forming polypeptides such as TEL-SAM and Aβ42 in Escherichia coli cells. Further and most importantly, the solubility of all poorly soluble target proteins examined was greatly elevated by their corresponding highly soluble hyper-acidic fusion cognates when they were co-expressed, in despite of a concomitant compromise of the cognates' solubility. The extent of such a solubility enhancement appeared to be in parallel with the ratio of the levels of co-expressed hyper-acidic fusion cognate and target protein. The hyper-acidic fusion cognates might function as intermolecular solubilizing effectors to prevent aggregation of the target proteins, and a plausible model for interpreting these results is also proposed.     

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.     

Interactive construction of residue-based diagrams of proteins: the RbDe web service

We announce the Residue-based Diagram Editor (RbDe) web service that allows online construction of residue-based diagrams and the creation of stored diagram libraries. The service has been tuned for the construction of snake-like diagrams (for transmembrane proteins) but can be used to render any protein for which defined secondary structure data or hypotheses are available. RbDe is freely available through the Internet from our web site: http://transport.physbio. mssm.edu/rbde/RbDe.html. Licenses for intranet uses can be obtained upon request.     

BRICHOS domain associated with lung fibrosis, dementia and cancer--a chaperone that prevents amyloid fibril formation?

The BRICHOS domain was initially defined from sequence alignments of the Bri protein associated with familial dementia, chondromodulin associated with chondrosarcoma and surfactant protein C precursor (proSP-C) associated with respiratory distress syndrome and interstitial lung disease (ILD). Today BRICHOS has been found in 12 protein families. Mutations in the Bri2 and proSP-C genes result in familial dementia and ILD, respectively, and both these conditions are associated with amyloid formation. Amyloid is of great medical relevance as it is found in several major incurable diseases, like Alzheimer's and Parkinson's disease and diabetes mellitus. Work on recombinant BRICHOS domains and transfected cells indicate that BRICHOS is a chaperone domain that, during biosynthesis, binds to precursor protein regions with high β-sheet propensities, thereby preventing them from amyloid formation. Regions prone to form β-sheets are present in all BRICHOS-containing precursor proteins and are probably eventually released by proteolytic cleavage, generating different peptides with largely unknown bioactivities. Recombinant BRICHOS domains from Bri2 and proSP-C have been found to efficiently prevent SP-C, the amyloid β-peptide associated with Alzheimer's disease, and medin, found in aortic amyloid, from forming amyloid fibrils. The data collected so far on BRICHOS raise several interesting topics for further research: (a) amyloid formation is a potential threat for many more proteins than the ones recognized so far in amyloid diseases; (b) amyloid formation of widely different peptides involves intermediate(s) that are recognized by the BRICHOS domain, suggesting that they have distinct structural similarities; and (c) the BRICHOS domain might be harnessed in therapeutic strategies against amyloid diseases.     

Alternative packing modes leading to amyloid polymorphism in five fragments studied with molecular dynamics

Amyloid aggregates have been implicated in the pathogenesis of diseases such as type 2 diabetes, Alzheimer's, Parkinson's, and prion disease. Recently determined microcrystal structures of several short peptide segments derived from fibril-forming proteins revealed coexistence of alternative aggregation modes (amyloid polymorphism) formed by the same segment. This polymorphism may help in understanding the influence of the side chain packing on the amyloid stability. Here we use molecular dynamics (MD) simulation to analyze the stability of five pairs of polar and nonpolar polymorphic oligomers. MD simulation shows polymorphs with steric zipper interface containing large polar and/or aromatic side chains (GNNQQNY, and NNQNTF) are more stable than steric zipper interfaces made of small or hydrophobic residues (SSTNGVG, VQIVYK, and MVGGVV). Several geometric analyses revealed that larger sheet to sheet interface of the dry steric zipper through polar Q/N rich side chains holds the sheets together. Mutant simulations (Q/N→G) show substitutions with glycine disrupt the steric zipper, leading to unstable oligomers. Stability of Q/N rich oligomers was found to result from the large average number of hydrogen bonds. The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method reports the nonpolar component of free energy to be favorable, while electrostatic solvation is unfavorable for β-sheet association. Knowledge of structural properties of these fibrils might be useful for developing therapeutic agents against amyloidoses as well as for developing biomaterials. ? 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 131-144, 2012.     

Towards a pharmacophore for amyloid

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.