Use of domain specific ligands to study urea-induced unfolding of bovine serum albumin

Urea-induced structural transitions in different domains of bovine serum albumin (BSA) were studied fluorometrically using domain specific ligands; chloroform, bilirubin, and diazepam. Urea denaturation of BSA showed a two-step, three-state transition with the accumulation of an intermediate around 4.8-5.2 M urea. During first transition (0-5.0 M urea), a continuous decrease (starting from 1.0 M urea) in diazepam (a ligand for domain III) binding and a late (from 3.0 M urea onward) decrease in chloroform (a ligand primarily for domain I) binding suggested major conformational changes in domain III and partial but significant loss of native conformation in domain I prior to intermediate formation. Absence of any decrease in bilirubin (a ligand for domain II) binding up to 4.5 M urea indicated non-involvement of domain II in the unfolding of BSA in this region. However, decrease in bilirubin binding during second transition reflected the unfolding of domain II and its separation from domain I.     

Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIalpha (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.     

Colipase: structure and interaction with pancreatic lipase

Colipase is a small protein cofactor needed by pancreatic lipase for the efficient dietary lipid hydrolysis. It binds to the C-terminal, non-catalytic domain of lipase, thereby stabilising an active conformation and considerably increasing the overall hydrophobic binding site. Structural studies of the complex and of colipase alone have clearly revealed the functionality of its architecture. Interestingly, a structural analogy has recently been discovered between colipase and a domain in a developmental protein (Dickkopf), based on sequence analogy and homology modeling. Whether this structural analogy implies a common function (lipid interaction) remains to be clarified. Structural analogies have also been recognised between the pancreatic lipase C-terminal domain, the N-terminal domains of lipoxygenases and the C-terminal domain of alpha-toxin. These non-catalytic domains in the latter enzymes are important for interaction with membranes. It has not been established if these domains are also involved in eventual protein cofactor binding as is the case for pancreatic lipase.     

Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.     

Improved stability of a protein vaccine through elimination of a partially unfolded state

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.     

The first epidermal growth factor-like domain of the low-density lipoprotein receptor contains a noncanonical calcium binding site

Removal of cholesterol-containing particles from the circulation is mediated by the low-density lipoprotein (LDL) receptor. Upon ligand binding, the receptor-ligand complex is endocytosed, and the ligand is released. The important biological role of the LDL receptor (LDLR) has been highlighted by the identification of more than 400 LDLR mutations that are associated with familial hypercholesterolemia. The extracellular region of the LDLR is modular in nature and principally comprises multiple copies of ligand binding, epidermal growth factor-like (EGF), and YWTD-type domains. This report describes characterization of the calcium binding properties of the tandem pair of EGF domains. While only the C-terminal EGF module contains the consensus sequence associated with calcium binding, a noncanonical calcium binding site in the N-terminal domain has been revealed using solution NMR spectroscopy. The calcium dissociation constants for the N- and C-terminal sites have been measured under physiologically relevant pH and ionic strength conditions using a combination of solution NMR, intrinsic protein fluorescence, and chromophoric chelator methods to be approximately 50 microM and approximately 10-20 microM, respectively. Identification of the novel calcium binding motif in LDLR sequences from other species suggests that it may confer specificity within the LDLR gene family. Comparison of the K(d) for the C-terminal site with the calcium concentration in late vesicles indicates that the binding properties of this module may be tuned to titrate upon endocytosis of the LDL receptor-ligand complex, and thus calcium binding may play a role in the ligand dissociation process.     

The structure and dynamics of tandem WW domains in a negative regulator of notch signaling, Suppressor of deltex

WW domains mediate protein recognition, usually though binding to proline-rich sequences. In many proteins, WW domains occur in tandem arrays. Whether or how individual domains within such arrays cooperate to recognize biological partners is, as yet, poorly characterized. An important question is whether functional diversity of different WW domain proteins is reflected in the structural organization and ligand interaction mechanisms of their multiple domains. We have determined the solution structure and dynamics of a pair of WW domains (WW3-4) from a Drosophila Nedd4 family protein called Suppressor of deltex (Su(dx)), a regulator of Notch receptor signaling. We find that the binding of a type 1 PPPY ligand to WW3 stabilizes the structure with effects propagating to the WW4 domain, a domain that is not active for ligand binding. Both WW domains adopt the characteristic triple-stranded beta-sheet structure, and significantly, this is the first example of a WW domain structure to include a domain (WW4) lacking the second conserved Trp (replaced by Phe). The domains are connected by a flexible linker, which allows a hinge-like motion of domains that may be important for the recognition of functionally relevant targets. Our results contrast markedly with those of the only previously determined three-dimensional structure of tandem WW domains, that of the rigidly oriented WW domain pair from the RNA-splicing factor Prp40. Our data illustrate that arrays of WW domains can exhibit a variety of higher order structures and ligand interaction mechanisms.     

Identification of human transferrin-binding sites within meningococcal transferrin-binding protein B.

Transferrin-binding protein B (TbpB) from Neisseria meningitidis binds human transferrin (hTf) at the surface of the bacterial cell as part of the iron uptake process. To identify hTf binding sites within the meningococcal TbpB, defined regions of the molecule were produced in Escherichia coli by a translational fusion expression system and the ability of the recombinant proteins (rTbpB) to bind peroxidase-conjugated hTf was characterized by Western blot and dot blot assays. Both the N-terminal domain (amino acids [aa] 2 to 351) and the C-terminal domain (aa 352 to 691) were able to bind hTf, and by a peptide spot synthesis approach, two and five hTf binding sites were identified in the N- and C-terminal domains, respectively. The hTf binding activity of three rTbpB deletion variants constructed within the central region (aa 346 to 543) highlighted the importance of a specific peptide (aa 377 to 394) in the ligand interaction. Taken together, the results indicated that the N- and C-terminal domains bound hTf approximately 10 and 1000 times less, respectively, than the full-length rTbpB (aa 2 to 691), while the central region (aa 346 to 543) had a binding avidity in the same order of magnitude as the C-terminal domain. In contrast with the hTf binding in the N-terminal domain, which was mediated by conformational epitopes, linear determinants seemed to be involved in the hTf binding in the C-terminal domain. The host specificity for transferrin appeared to be mediated by the N-terminal domain of the meningococcal rTbpB rather than the C-terminal domain, since we report that murine Tf binds to the C-terminal domain. Antisera raised to both N- and C-terminal domains were bactericidal for the parent strain, indicating that both domains are accessible at the bacterial surface. We have thus identified hTf binding sites within each domain of the TbpB from N. meningitidis and propose that the N- and C-terminal domains together contribute to the efficient binding of TbpB to hTf with their respective affinities and specificities for determinants of their ligand.     

Identification and kinetic analysis of the interaction between Nck-2 and DOCK180

Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain. We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.     

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

βPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of βPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of βPIX, which is important for its physiological functions. We have solved the crystal structure of the βPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The βPIX CC forms a 76-Å-long parallel CC trimer. Despite the fact that the βPIX CC exposes three PDZ binding motifs in the C-termini, the βPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive β-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of βPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the βPIX trimer. The structure shows canonical class I PDZ binding with a β-sheet interaction extending to position − 6 of βPIX. The βB-βC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the β-sheet interaction and to accommodate the bulky side chain of Trp − 5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of βPIX CC and Shank PDZ.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

含磷酸结合口袋的BRCT结构域功能位点预测

BRCT( BRCA1 C-terminus)是真核生物DNA损伤修复系统重要的信号传导和蛋白靶向结构域.为了探讨含磷酸结合口袋的BRCT与磷酸化配体结合的机制,对XRCC1 BRCT1、PTIP BRCT4、ECT2 BRCT1和TopBP1BRCT1进行了结构保守性和表面静电势分析.结果显示,4个BRCT的磷酸结合口袋周围所存在的结构保守并带正电势的沟槽很可能是其功能位点,并且类似的沟槽在含磷酸结合口袋的BRCT中普遍存在.沟槽两侧及底部均带有极性氨基酸残基,两侧带正电荷,而底部疏水.这说明沟槽与配体的结合以静电和疏水相互作用为主.沟槽主要位于单个BRCT中,而且4个BRCT的沟槽在形状和电荷分布上都不同,说确明BRCT配体特异性主要由单个BRCT决定.磷酸结合口袋位于沟槽中心,说明沟槽可能同时结合磷酸化残基的N端和C端附近残基.     

Insight into the structure and function of the transferrin receptor from Neisseria meningitidis using microcalorimetric techniques

The transferrin receptor of Neisseria meningitidis is composed of the transmembrane protein TbpA and the outer membrane protein TbpB. Both receptor proteins have the capacity to independently bind their ligand human transferrin (htf). To elucidate the specific role of these proteins in receptor function, isothermal titration calorimetry was used to study the interaction between purified TbpA, TbpB or the entire receptor (TbpA + TbpB) with holo- and apo-htf. The entire receptor was shown to contain a single high affinity htf-binding site on TbpA and approximately two lower affinity binding sites on TbpB. The binding sites appear to be independent. Purified TbpA was shown to have strong ligand preference for apo-htf, whereas TbpA in the receptor complex with TbpB preferentially binds the holo form of htf. The orientation of the ligand specificity of TbpA toward holo-htf is proposed to be the physiological function of TbpB. Furthermore, the thermodynamic mode of htf binding by TbpB of isotypes I and II was shown to be different. A protocol for the generation of active, histidine-tagged TbpB as well as its individual N- and C-terminal domains is presented. Both domains are shown to strongly interact with each other, and isothermal titration calorimetry and circular dichroism experiments provide clear evidence for this interaction causing conformational changes. The N-terminal domain of TbpB was shown to be the site of htf binding, whereas the C-terminal domain is not involved in binding. Furthermore, the interactions between TbpA and the different domains of TbpB have been demonstrated.     

Calorimetric analysis of the Ca(2+)-binding betagamma-crystallin homolog protein S from Myxococcus xanthus: intrinsic stability and mutual stabilization of domains.

The betagamma-crystallin superfamily consists of a class of homologous two-domain proteins with Greek-key fold. Protein S, a Ca(2+)-binding spore-coat protein from the soil bacterium Myxococcus xanthus exhibits a high degree of sequential and structural homology with gammaB-crystallin from the vertebrate eye lens. In contrast to gammaB-crystallin, which undergoes irreversible aggregation upon thermal unfolding, protein S folds reversibly and may therefore serve as a model in the investigation of the thermodynamic stability of the eye-lens crystallins. The thermal denaturation of recombinant protein S (PS) and its isolated domains was studied by differential scanning calorimetry in the absence and in the presence of Ca(2+) at varying pH. Ca(2+)-binding leads to a stabilization of PS and its domains and increases the cooperativity of their equilibrium unfolding transitions. The isolated N-terminal and C-terminal domains (NPS and CPS) obey the two-state model, independent of the pH and Ca(2+)-binding; in the case of PS, under all conditions, an equilibrium intermediate is populated. The first transition of PS may be assigned to the denaturation of the C-terminal domain and the loss of domain interactions, whereas the second one coincides with the denaturation of the isolated N-terminal domain. At pH 7.0, in the presence of Ca(2+), where PS exhibits maximal stability, the domain interactions at 20 degrees C contribute 20 kJ/mol to the overall stability of the intact protein.     

Convergent and divergent ligand specificity among PDZ domains of the LAP and zonula occludens (ZO) families

We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight the importance of site(-1), which interacts with the penultimate residue of ligand C termini. Site(-1) was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1 (accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity within site(-1) greatly influences the range of potential biological partners and functions that can be associated with each protein. These findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.     

Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide

Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.     

A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.     

Expression, Purification, and Biophysical Characterization of the BRCT Domain of Human DNA Ligase IIIα

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIα (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837–922 of ligase IIIα) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, ¹⁵N-labeled and ¹³C/¹⁵N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIα activity.