Pairs Of Hydrophobic Residues Research Articles

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

The information on the 3D structure of a protein including its folding mechanism is encoded in its amino acid sequence. A β-trefoil protein is well known to have a remarkable 3D structure property, that is, the pseudo three-fold symmetry without clear hydrophobic packing. It is interesting to investigate how information on the folding mechanism to form such a topology is encoded in the amino acid sequence of a protein. In this study, analyses based on inter-residue average distance statistics and the conservation of hydrophobic residues are performed for sequences of 26 β-trefoil proteins to identify significant sites for the initial folding. Results are compared with the native 3D structures. The conserved hydrophobic residues are defined by multiple sequence alignment based on the 3D structures. It is confirmed that a conserved hydrophobic residue is always located in a β-strand. In particular, β-strands 5 and 6 are significant for the initial folding from the analyses based on the inter-residue average distance statistics. These results coincide well with the experimental data obtained so far for folding of some of the β-trefoil proteins. It is also confirmed that the conserved hydrophobic residues defined in this study contribute to form hydrophobic packing in β-trefoil proteins in general. Twelve conserved hydrophobic residue pairs are almost always observed to form packing in the 26 β-trefoil proteins from different superfamilies. We elucidate how the conserved hydrophobic residues in β-strands 5 and 6 contribute to the initial stage of folding of a β-trefoil protein. The common packing of the 12 conserved hydrophobic residue pairs are significant to form the whole β-trefoil fold structure.

Hydophobic Guided Protein Folding

Protein folding simulations have the potential to help us figure out the problem of protein folding. However, the highly frustrated energy landscapes of protein folding limits the use of these techniques. Hence, many new algorithms are developed each year aiming at improving sampling efficiency, producing results in shorter times. We have developed and tested an algorithm using GPU technology that allows us to fold several proteins within weeks of computational usage.Our work extends on the principles of protein folding through zipping and assembly. In this view, local events such as helix formation happen first (zipping), and non-local ones later (assembly). The assembly step is slow: in order to form native like contacts the residues involved have to first find each other. Our algorithm takes advantage of the fact that proteins form hydrophobic cores to guide the assembly step. We accelerate the process by adding restraints in our simulations that brings pairs of hydrophobic residues together, favoring the formation of native-like contacts. Since not all hydrophobic pairs will lead to native-like contacts we use a Hamiltonian and Temperature Replica exchange algorithm (H,T-REMD), that allows us to modulate the strengths of these restraints. Thus, the restraints that the underlying molecular force field favors are enforced, where as the ones that are not favored are turned off.This method allows us to jump between different sets of contacts easily, sampling many different topologies and limiting the amount of sampling that is required in between. In this way we have been able to get native-like structures for a small set of proteins. During my presentation I will show how the method works and how successful it is in a small set of proteins.

Viral Infection Controlled by a Calcium-Dependent Lipid-Binding Module in ALIX

ALIX plays a role in nucleocapsid release during viral infection, as does lysobisphosphatidic acid (LBPA). However, the mechanism remains unclear. Here we report that LBPA is recognized within an exposed site in ALIX Bro1 domain predicted by MODA, an algorithm for discovering membrane-docking areas in proteins. LBPA interactions revealed a strict requirement for a structural calcium tightly bound near the lipid interaction site. Unlike other calcium- and phospholipid-binding proteins, the all-helical triangle-shaped fold of the Bro1 domain confers selectivity for LBPA via a pair of hydrophobic residues in a flexible loop, which undergoes a conformational change upon membrane association. Both LBPA and calcium binding are necessary for endosome association and virus infection, as are ALIX ESCRT binding and dimerization capacity. We conclude that LBPA recruits ALIX onto late endosomes via the calcium-bound Bro1 domain, triggering a conformational change in ALIX to mediate the delivery of viral nucleocapsids to the cytosol during infection.

Analyses of the general rule on residue pair frequencies in local amino acid sequences of soluble, ordered proteins

The amino acid sequences of soluble, ordered proteins with stable structures have evolved due to biological and physical requirements, thus distinguishing them from random sequences. Previous analyses have focused on extracting the features that frequently appear in protein substructures, such as α-helix and β-sheet, but the universal features of protein sequences have not been addressed. To clarify the differences between native protein sequences and random sequences, we analyzed 7368 soluble, ordered protein sequences, by inspecting the observed and expected occurrences of 400 amino acid pairs in local proximity, up to 10 residues along the sequence in comparison with their expected occurrence in random sequence. We found the trend that the hydrophobic residue pairs and the polar residue pairs are significantly decreased, whereas the pairs between a hydrophobic residue and a polar residue are increased. This trend was universally observed regardless of the secondary structure content but was not observed in protein sequences that include intrinsically disordered regions, indicating that it can be a general rule of protein foldability. The possible benefits of this rule are discussed from the viewpoints of protein aggregation and disorder, which are both caused by low-complexity regions of hydrophobic or polar residues.

A Tandem Di-hydrophobic Motif Mediates Clathrin-dependent Endocytosis via Direct Binding to the AP-2 ασ2 Subunits

Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.

Protein β-Sheet Nucleation Is Driven by Local Modular Formation

Despite its central role in the protein folding process, the specific mechanism(s) behind beta-sheet formation has yet to be determined. For example, whether the nucleation of beta-sheets, often containing strands separated in sequence by many residues, is local or not remains hotly debated. Here, we investigate the initial nucleation step of beta-sheet formation by performing an analysis of the smallest beta-sheets in a non-redundant dataset on the grounds that the smallest sheets, having undergone little growth after nucleation, will be enriched for nucleating characteristics. We find that the residue propensities are similar for small and large beta-sheets as are their interstrand pairing preferences, suggesting that nucleation is not primarily driven by specific residues or interacting pairs. Instead, an examination of the structural environments of the two-stranded sheets shows that virtually all of them are contained in single, compact structural modules, or when multiple modules are present, one or both of the chain termini are involved. We, therefore, find that beta-nucleation is a local phenomenon resulting either from sequential or topological proximity. We propose that beta-nucleation is a result of two opposite factors; that is, the relative rigidity of an associated folding module that holds two stretches of coil close together in topology coupled with sufficient chain flexibility that enables the stretches of coil to bring their backbones in close proximity. Our findings lend support to the hydrophobic zipper model of protein folding (Dill, K. A., Fiebig, K. M., and Chan, H. S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1942-1946). Implications for protein folding are discussed.

Iterative Assembly of Protein Fragments

MASH (Magical Assembly of Sheets and Helices) is a computational method for iterative assembly of protein secondary structure fragments into native-like tertiary structures. For a given pair of fragments, we select a pair of hydrophobic residues (in the case of helix assembly) or an H-bond donor/acceptor pair (in the case of beta strands). We then position the two fragments so that the selected residues are in close proximity to one another. Finally, we attempt to connect the two fragments with a robotics-based loop-closure algorithm.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

The Mutations SER2117→LEU, and TRP/TRP2063/64→MET/PHE in the Hydrophobic Spikes of the Factor V C2 Domain Lead to Tenacious Phospholipid Binding and Suggest a Mechanism through Which Phosphatidylserine Activates the Prothrombinase Complex.

Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

The structural basis of novel endosome anchoring activity of KIF16B kinesin

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.

Switch interactions control energy frustration and multiple flagellar filament structures

Bacterial flagellar filament is a macromolecular assembly consisting of a single protein, flagellin. Bacterial swimming is controlled by the conformational transitions of this filament between left- and right-handed supercoils induced by the flagellar motor torque. We present a massive molecular dynamics simulation that was successful in constructing the atomic-level supercoil structures consistent with various experimental data and further in elucidating the detailed underlying molecular mechanisms of the polymorphic supercoiling. We have found that the following three types of interactions are keys to understanding the supercoiling mechanism. "Permanent" interactions are always maintained between subunits in the various supercoil structures. "Sliding" interactions are formed between variable hydrophilic or hydrophobic residue pairs, allowing intersubunit shear without large change in energy. The formation and breakage of "switch" interactions stabilize inter- and intrasubunit interactions, respectively. We conclude that polymorphic supercoiling is due to the energy frustration between them. The transition between supercoils is achieved by a "transform and relax" mechanism: the filament structure is geometrically transformed rapidly and then slowly relaxes to energetically metastable states by rearranging interactions.

Asparagine Deamidation Perturbs Antigen Presentation on Class II Major Histocompatibility Complex Molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Role of medium- and long-range interactions in discriminating globular and membrane proteins

The analysis of inter-residue interactions in protein structures provides considerable insight to understand their folding and stability. We have previously analyzed the role of medium- and long-range interactions in the folding of globular proteins. In this work, we study the distinct role of such interactions in the three-dimensional structures of membrane proteins. We observed a higher number of long-range contacts in the termini of transmembrane helical (TMH) segments, implying their role in the stabilization of helix–helix interactions. The transmembrane strand (TMS) proteins are having appreciably higher long-range contacts than that in all-β class of globular proteins, indicating closer packing of the strands in TMS proteins. The residues in membrane spanning segments of TMH proteins have 1.3 times higher medium-range contacts than long-range contacts whereas that of TMS proteins have 14 times higher long-range contacts than medium-range contacts. Residue-wise analysis indicates that in TMH proteins, the residues Cys, Glu, Gly, Pro, Gln, Ser and Tyr have higher long-range contacts than medium-range contacts in contrast with all-α class of globular proteins. The charged residue pairs have higher medium-range contacts in all-α proteins, whereas hydrophobic residue pairs are dominant in TMH proteins. The information on the preference of residue pairs to form medium-range contacts has been successfully used to discriminate the TMH proteins from all-α proteins. The statistical significance of the results obtained from the present study has been verified using randomized structures of TMH and TMS protein templates.

Glassy dynamics of protein folding.

A coarse-grained model of a random polypeptide chain, with only discrete torsional degrees of freedom and Hookean springs connecting pairs of hydrophobic residues is shown to display stretched exponential relaxation under Metropolis dynamics at low temperatures with the exponent beta approximately 1/4, in agreement with the best experimental results. The time dependent correlation functions for fluctuations about the native state, computed in the Gaussian approximation for real proteins, have also been found to have the same functional form. Our results indicate that the energy landscape exhibits universal features over a very large range of energies and is relatively independent of the specific dynamics.

Dissipative dynamics and the statistics of energy states of a Hookean model for protein folding

A generic model of a random polypeptide chain, with discrete torsional degrees of freedom and Hookean spring connecting pairs of hydrophobic residues, reproduces the energy probability distribution of real proteins over a very large range of energies. We show that this system with harmonic interactions, under dissipative dynamics driven by random noise, leads to a distribution of energy states obeying a modified one-dimensional Ornstein–Uhlenbeck process and giving rise to the so-called Wigner distribution. A tunably fine- or coarse-grained sampling of the energy landscape yields a family of distributions for the energies and energy spacings.

Natural processing sites for human cathepsin E and cathepsin D in tetanus toxin: implications for T cell epitope generation.

Cathepsin E is an aspartic proteinase that has been implicated frequently in Ag processing for presentation on class II MHC molecules, but no information exists on its cleavage specificity within Ags in relation to known T cell epitopes. We have analyzed the processing by cathepsin E of a large C-terminal domain of tetanus toxin (residues 872-1315), and we have compared the processing products with those liberated by cathepsin D, a related aspartic proteinase also thought to be involved in class II MHC-restricted Ag processing. Processing products were analyzed by N-terminal Edman degradation and mass spectrometry following reverse-phase HPLC separation of peptides. A total of 28 cleavage sites was identified, 11 of which were recognized by both cathepsins E and D. Most, although not all, sites were between pairs of hydrophobic residues and were located within the 200-amino-acid C terminal region known to contain several human T cell epitopes. Previously described T cell epitopes, for example, between residues 1273 and 1284, were flanked by cathepsin E and D cleavage sites. These data are consistent with an important role for cathepsins E and/or D in Ag processing in the human immune system.

Interactions between hydrophobic side chains within α‐helices

The thermodynamic basis of helix stability in peptides and proteins is a topic of considerable interest. Accordingly, we have computed the interactions between side chains of all hydrophobic residue pairs and selected triples in a model helix, using Boltzmann-weighted exhaustive modeling. Specifically, all possible pairs from the set Ala, Cys, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val were modeled at spacings of (i, i + 2), (i, i + 3), and (i, i + 4) in the central turn of a model poly-alanyl alpha-helix. Significant interactions--both stabilizing and destabilizing-- were found to occur at spacings of (i, i + 3) and (i, i + 4), particularly in side chains with rings (i.e., Phe, Tyr, Trp, and His). In addition, modeling of leucine triples in a helix showed that the free energy can exceed the sum of pairwise interactions in certain cases. Our calculated interaction values both rationalize recent experimental data and provide previously unavailable estimates of the constituent energies and entropies of interaction.

Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10).

Carboxypeptidase E (CPE), a peptide hormone-processing enzyme, is present within secretory granules in both a soluble form and a form which is membrane-bound at pH 5.5 but soluble at neutral pH. Antisera raised against a peptide corresponding to the predicted COOH-terminus of CPE bind to the membrane-associated form of CPE but not to the soluble form. This COOH-terminal region is predicted to form an amphiphilic alpha-helix, containing several pairs of hydrophobic residues separated by hydrophilic residues. Synthetic COOH-terminal peptides 11-24 residues in length are able to bind to bovine pituitary membranes and can be extracted by conditions that extract the membrane-bound form of CPE. The influence of pH on the membrane binding of a 21-residue COOH-terminal peptide is similar to the membrane binding of CPE: at pH values less than 6 the majority of the peptide is membrane-bound, while at pH values above 8 less than 20% is membrane-bound. Both the 21-residue COOH-terminal peptide and the purified membrane form of CPE, but not the soluble form, partition into Triton X-114 only at low pH (pH less than 6). Combined polar and hydrophobic interactions of the COOH-terminal peptide appear to be responsible for the reversible, pH-dependent association of CPE with membranes.