Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine

The adaptor protein Hop mediates the association of the molecular chaperones Hsp70 and Hsp90. The TPR1 domain of Hop specifically recognizes the C-terminal heptapeptide of Hsp70 while the TPR2A domain binds the C-terminal pentapeptide of Hsp90. Both sequences end with the motif EEVD. The crystal structures of the TPR-peptide complexes show the peptides in an extended conformation, spanning a groove in the TPR domains. Peptide binding is mediated by electrostatic interactions with the EEVD motif, with the C-terminal aspartate acting as a two-carboxylate anchor, and by hydrophobic interactions with residues upstream of EEVD. The hydrophobic contacts with the peptide are critical for specificity. These results explain how TPR domains participate in the ordered assembly of Hsp70-Hsp90 multichaperone complexes.     

Binding surface mapping of intra- and interdomain interactions among hHR23B,ubiquitin, and polyubiquitin binding site 2 of S5a

hHR23B is the human homologue of the yeast protein RAD23 and is known to participate in DNA repair by stabilizing xeroderma pigmentosum group C protein. However, hHR23B and RAD23 also have many important functions related to general proteolysis. hHR23B consists of N-terminal ubiquitin-like (UbL), ubiquitin association 1 (UBA1), xeroderma pigmentosum group C binding, and UBA2 domains. The UBA domains interact with ubiquitin (Ub) and inhibit the assembly of polyubiquitin. On the other hand, the UbL domain interacts with the poly-Ub binding site 2 (PUbS2) domain of the S5a protein, which can carry polyubiquitinated substrates into the proteasome. We calculated the NMR structure of the UbL domain of hHR23B and determined binding surfaces of UbL and Ub to UBA1, UBA2, of hHR23B and PUbS2 of S5a by using chemical shift perturbation. Interestingly, the surfaces of UbL and Ub that bind to UBA1, UBA2, and PUbS2 are similar, consisting of five beta-strands and their connecting loops. This is the first report that an intramolecular interaction between UbL and UBA domains is possible, and this interaction could be important for the control of proteolysis by hHR23B. The binding specificities of UbL and Ub for PUbS1, PUbS2, and general ubiquitin-interacting motifs, which share the LALA motif, were evaluated. The UBA domains bind to the surface of Ub including Lys-48, which is required for multiubiquitin assembly, possibly explaining the observed inhibition of multiubiquitination by hHR23B. The UBA domains bind to UbL through electrostatic interactions supported by hydrophobic interactions and to Ub mainly through hydrophobic interactions supported by electrostatic interactions.     

Mechanical unbinding of abeta peptides from amyloid fibrils

Using the experimental structures of Abeta amyloid fibrils and all-atom molecular dynamics, we study the force-induced unbinding of Abeta peptides from the fibril. We show that the mechanical dissociation of Abeta peptides is highly anisotropic and proceeds via different pathways when force is applied in parallel or perpendicular direction with respect to the fibril axis. The threshold forces associated with lateral unbinding of Abeta peptides exceed those observed during the mechanical dissociation along the fibril axis. In addition, Abeta fibrils are found to be brittle in the lateral direction of unbinding and soft along the fibril axis. Lateral mechanical unbinding and the unbinding along the fibril axis load different types of fibril interactions. Lateral unbinding is primarily determined by the cooperative rupture of fibril backbone hydrogen bonds. The unbinding along the fibril axis largely depends on the interpeptide Lys-Asp electrostatic contacts and the hydrophobic interactions formed by the Abeta C terminal. Due to universality of the amyloid beta structure, the anisotropic mechanical dissociation observed for Abeta fibrils is likely to be applicable to other amyloid assemblies. The estimates of equilibrium forces required to dissociate Abeta peptide from the amyloid fibril suggest that these supramolecular structures are mechanically stronger than most protein domains.     

Molecular dynamics simulations of a calmodulin-peptide complex in solution

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 A(2) approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 A(2), comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to peptide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands.

The receptor, a maltose/maltooligosaccharide-binding protein, has been found to be an excellent system for the study of molecular recognition because its polar and nonpolar binding functions are segregated into two globular domains. The X-ray structures of the "closed" and "open" forms of the protein complexed with maltose and maltotetraitol have been determined. These sugars have approximately 3 times more accessible polar surface (from OH groups) than nonpolar surface (from small clusters of sugar ring CH bonds). In the closed structures, the oligosaccharides are buried in the groove between the two domains of the protein and bound by extensive hydrogen bonding interactions of the OH groups with the polar residues confined mostly in one domain and by nonpolar interactions of the CH clusters with four aromatic residues lodged in the other domain. Substantial contacts between the sugar hydroxyls and aromatic residues are also formed. In the open structures, the oligosaccharides are bound almost exclusively in the domain rich in aromatic residues. This finding, along with the analysis of buried surface area due to complex formations in the open and closed structures, supports a major role for nonpolar interactions in initial ligand binding even when the ligands have significantly greater potential for highly specific polar interactions.     

Investigation at residue level of the early steps during the assembly of two proteins into supramolecular objects

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.     

Molecular Dynamics Simulations of a Calmodulin-peptide Complex in Solution

Abstract

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 Ų, approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 ·A², comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to pep- tide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

The excised heat-shock domain of alphaB crystallin is a folded, proteolytically susceptible trimer with significant surface hydrophobicity and a tendency to self-aggregate upon heating

The lens protein, alpha-crystallin, is a molecular chaperone that prevents the thermal aggregation of other proteins. The C-terminal domain of this protein (homologous to domains present in small heat-shock proteins) is implicated in chaperone function, although the domain itself has been reported to show no chaperone activity. Here, we show that the domain can be excised out of the intact alphaB polypeptide and recovered directly in pure form through the transfer of CNBr digests of whole lens homogenates into urea-containing buffer, followed by dialysis-based refolding of digests under acidic conditions and a single gel-filtration purification step. The folded (beta sheet) domain thus obtained is found to be (a) predominantly trimeric, and to display (b) significant surface hydrophobicity, (c) a marked tendency to undergo degradation, and (d) a tendency to aggregate upon heating, and on exposure to UV light. Thus, the twin 'chaperone' features of multimericity and surface hydrophobicity are clearly seen to be insufficient for this domain to function as a chaperone. Since alpha-crystallin interacts with its substrates through hydrophobic interactions, the hydrophobicity of the excised domain indicates that separation of domains may regulate function; at the same time, the fact is also highlighted that surface hydrophobicity is a liability in a chaperone since heating strengthens hydrophobic interactions and can potentially promote self-aggregation. Thus, it would appear that the role of the N-terminal domain in alpha-crystallin is to facilitate the creation of a porous, hollow structural framework of >/=24 subunits in which solubility is effected through increase in the ratio of exposed surface area to buried volume. Trimers of interacting C-terminal domains anchored to this superstructure, and positioned within its interior, might allow hydrophobic surfaces to remain accessible to substrates without compromising solubility.     

The regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.     

Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin

The lipid second messenger diacylglycerol acts by binding to the C1 domains of target proteins, which translocate to cell membranes and are allosterically activated. Here we report the crystal structure at 3.2 A resolution of one such protein, beta2-chimaerin, a GTPase-activating protein for the small GTPase Rac, in its inactive conformation. The structure shows that in the inactive state, the N terminus of beta2-chimaerin protrudes into the active site of the RacGAP domain, sterically blocking Rac binding. The diacylglycerol and phospholipid membrane binding site on the C1 domain is buried by contacts with the four different regions of beta2-chimaerin: the N terminus, SH2 domain, RacGAP domain, and the linker between the SH2 and C1 domains. Phospholipid binding to the C1 domain triggers the cooperative dissociation of these interactions, allowing the N terminus to move out of the active site and thereby activating the enzyme.     

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.     

Packing forces in ribonuclease crystals

Packing in Ribonuclease A and Ribonuclease S crystals have been compared in order to determine the possible role of the precipitant on lattice contacts. Both proteins have similar tertiary structures, but they crystallize in different space groups depending on the precipitating agent. It is found that packing differs either by the number of nearest neighbours or by the size of surface areas buried in individual contacts. Ammonium sulfate seems to promote hydrophobic interactions with interfaces similar to those found in oligomeric proteins. Organic precipitants favour electrostatic interactions.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.     

Interdomain interactions in hinge-bending transitions.

BACKGROUND: The mechanisms that allow or constrain protein movement have not been understood. Here we study interdomain interactions in proteins to investigate hinge-bending motions. RESULTS: We find a limited number of salt bridges and hydrogen bonds at the interdomain interface, in both the "closed" and the "open" conformations. Consistently, analysis of 222 salt bridges in an independently selected database indicates that most salt bridges form within rather than between independently folding hydrophobic units. Calculations show that these interdomain salt bridges either destabilize or only marginally stabilize the closed conformation in most proteins. In contrast, the nonpolar buried surface area between the moving parts can be extensive in the closed conformations. However, when the nonpolar buried surface area is large, we find that at the interdomain interface in the open conformation it may be as large or larger than in the closed conformation. Hence, the energetic penalty of opening the closed conformation is overcome. Consistently, a large nonpolar surface area buried in the closed interdomain interface accompanies limited opening of the domains, yielding a larger interface. CONCLUSIONS: Short-range electrostatic interactions are largely absent between moving domains. Interdomain nonpolar buried surface area may be large in the closed conformation, but it is largely offset by the area buried in the open conformation. In such cases the opening of the domains appears to be relatively small. This may allow prediction of the extent of domain opening. Such predictions may have implications for the shape and size of the binding pockets in drug/protein design.