Avian pancreatic polypeptide fragments refold to native aPP conformation when combined in solution: a CD and VCD study

An equimolar mixture of avian pancreatic polypeptide (aPP) fragments aPP(1-11)-NH2 and Ac-aPP(12-36) had an electronic circular dichroism (ECD) spectrum that was similar to that of whole aPP in H2O and even more so in 30% (v/v) trifluoroethanol (TFE) in 15 mM Na2HPO4, but was different from the sum of the spectra of the individual fragments. The vibrational circular dichroism (VCD) spectrum of the combined fragments in 30% (v/v) TFE in 15 mM Na2HPO4 in D2O was also similar to that of the intact aPP and unlike the sum of the VCD spectra of the fragments. The interaction of these fragments is thus sufficient to support the conformation of whole aPP. This study demonstrates that VCD, in combination with ECD, is useful for the study of protein-protein interactions.     

A protein-RNA docking benchmark (I): nonredundant cases

We have developed a nonredundant protein-RNA docking benchmark dataset, which is derived from the available bound and unbound structures in the Protein Data Bank involving polypeptide and nucleic acid chains. It consists of nine unbound-unbound cases where both the protein and the RNA are available in the free form. The other 36 cases are of unbound-bound type where only the protein is available in the free form. The conformational change upon complex formation is calculated by a distance matrix alignment method, and based on that, complexes are classified into rigid, semi-flexible, and full flexible. Although in the rigid body category, no significant conformational change accompanies complex formation, the fully flexible test cases show large domain movements, RNA base flips, etc. The benchmark covers four major groups of RNA, namely, t-RNA, ribosomal RNA, duplex RNA, and single-stranded RNA. We find that RNA is generally more flexible than the protein in the complexes, and the interface region is as flexible as the molecule as a whole. The structural diversity of the complexes in the benchmark set should provide a common ground for the development and comparison of the protein-RNA docking methods. The benchmark can be freely downloaded from the internet.     

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions.     

TRIADs: a new class of proteins with a novel cysteine-rich signature.

Triad1 was recently identified as a nuclear RING finger protein, which is up-regulated during retinoic acid induced granulocytic differentiation of acute leukemia cells. Here we show that a cysteine-rich domain (C6HC), present in Triad1, is conserved in at least 24 proteins encoded by various eukaryotes. The C6HC consensus pattern C-x(4)-C-x(14-30)-C-x(1-4)-C-x(4)-C-x(2)-C-x(4)-H-x(4)-C defines this structure as the fourth family member of the zinc-binding RING, LIM, and LAP/PHD fingers. Strikingly, in 22 of 24 proteins the C6HC domain is flanked by two RING finger structures. We have termed the novel C6HC motif DRIL (double RING finger linked). The strong conservation of the larger tripartite TRIAD (two RING fingers and DRIL) structure indicates that the three subdomains are functionally linked and identifies a novel class of proteins.     

Structural coupling between FKBP12 and buried water

Globular proteins often contain structurally well-resolved internal water molecules. Previously, we reported results from a molecular dynamics study that suggested that buried water (Wat3) may play a role in modulating the structure of the FK506 binding protein-12 (FKBP12) (Park and Saven, Proteins 2005; 60:450-463). In particular, simulations suggested that disrupting a hydrogen bond to Wat3 by mutating E60 to either A or Q would cause a structural perturbation involving the distant W59 side chain, which rotates to a new conformation in response to the mutation. This effectively remodels the ligand-binding pocket, as the side chain in the new conformation is likely to clash with bound FK506. To test whether the protein structure is in effect modulated by the binding of a buried water in the distance, we determined high-resolution (0.92-1.29 A) structures of wild-type FKBP12 and its two mutants (E60A, E60Q) by X-ray crystallography. The structures of mutant FKBP12 show that the ligand-binding pocket is indeed remodeled as predicted by the substitution at position 60, even though the water molecule does not directly interact with any of the amino acids of the binding pocket. Thus, these structures support the view that buried water molecules constitute an integral, noncovalent component of the protein structure. Additionally, this study provides an example in which predictions from molecular dynamics simulations are experimentally validated with atomic precision, thus showing that the structural features of protein-water interactions can be reliably modeled at a molecular level.     

Chip-based protein-protein interaction studied by atomic force microscopy

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 10⁴ pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ～37% as compared with ～9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc.     

The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation

The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.     

Evaluation of different biological data and computational classification methods for use in protein interaction prediction

Protein–protein interactions play a key role in many biological systems. High‐throughput methods can directly detect the set of interacting proteins in yeast, but the results are often incomplete and exhibit high false‐positive and false‐negative rates. Recently, many different research groups independently suggested using supervised learning methods to integrate direct and indirect biological data sources for the protein interaction prediction task. However, the data sources, approaches, and implementations varied. Furthermore, the protein interaction prediction task itself can be subdivided into prediction of (1) physical interaction, (2) co‐complex relationship, and (3) pathway co‐membership. To investigate systematically the utility of different data sources and the way the data is encoded as features for predicting each of these types of protein interactions, we assembled a large set of biological features and varied their encoding for use in each of the three prediction tasks. Six different classifiers were used to assess the accuracy in predicting interactions, Random Forest (RF), RF similarity‐based k‐Nearest‐Neighbor, Naïve Bayes, Decision Tree, Logistic Regression, and Support Vector Machine. For all classifiers, the three prediction tasks had different success rates, and co‐complex prediction appears to be an easier task than the other two. Independently of prediction task, however, the RF classifier consistently ranked as one of the top two classifiers for all combinations of feature sets. Therefore, we used this classifier to study the importance of different biological datasets. First, we used the splitting function of the RF tree structure, the Gini index, to estimate feature importance. Second, we determined classification accuracy when only the top‐ranking features were used as an input in the classifier. We find that the importance of different features depends on the specific prediction task and the way they are encoded. Strikingly, gene expression is consistently the most important feature for all three prediction tasks, while the protein interactions identified using the yeast‐2‐hybrid system were not among the top‐ranking features under any condition. Proteins 2006. © 2006 Wiley‐Liss, Inc.     

The complex structure of calmodulin bound to a calcineurin peptide

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.