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The endoplasmic reticulum is a major compartment of protein biogenesis in the cell, dedicated to production of secretory, membrane and organelle proteins. The secretome has distinct structural and post-translational characteristics, since folding in the ER occurs in an environment that is distinct in terms of its ionic composition, dynamics and requirements for quality control. The folding machinery in the ER therefore includes chaperones and folding enzymes that introduce, monitor and react to disulfide bonds, glycans, and fluctuations of luminal calcium. We describe the major chaperone networks in the lumen and discuss how they have distinct modes of operation that enable cells to accomplish highly efficient production of the secretome. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins. This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

Homology modeling has been used to identify extra-chloroplastic enzymes that contain potential disulfide bonds. All of the higher plant fructose bisphosphatases and mitochondrial citrate synthases that have been tested to date, two glyceraldehyde-3-P dehydrogenases, two enolases and one lactate dehydrogenase, are redox-sensitive and may then be redox-regulated in vivo. Apparently, redox-sensitivity is not limited to the chloroplast.

With limited exceptions, proteins that account for the amyloidoses appear to be evolutionarily unrelated. Transthyretin is classified as having an "immunoglobulin-like" fold as found in light chain variable and constant domains. Thus, these amyloidogenic proteins have significant conformational similarity. In the absence of primary structure similarity sufficient to justify an inference of an evolutionary relationship, transthyretin is considered an analog of immunoglobulin domains having accrued the immunoglobulin-like fold by some form of convergent evolution of structure. Improvements in sequence comparison tools and strategies, coupled with recent logarithmic increases in the availability of primary structure data, now make it possible to suggest that transthyretin and immunoglobulins may have a common evolutionary origin. In addition, lactadherin, the medin fragment of which accounts for the most common form of human amyloid, also appears to be evolutionarily linked to transthyretin and immunoglobulins.

Many extracellular pathogenic bacteria colonize human or animal bodies through evasion of the host immune system, a process called host-pathogen interaction. What happens when other intruders try to invade the same host and try to establish themselves in the same niche is largely unknown. In one well-studied case, Pseudomonas aeruginosa is known to secrete the protein azurin as a weapon against such invaders as cancers, parasites and viruses. The production of such weapons by pathogenic bacteria could provide important insights into how a pathogen responds in the post-colonization state to impede other intruders for its own survival. Moreover, these molecules might find use in the pharmaceutical industry as next-generation therapeutics.

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (FALS), and approximately 25% of FALS cases are caused by mutations in superoxide dismutase-1 (SOD1). Mutant (MT) SOD1 kills motor neurons because of the mutant protein's toxicity; however, the basis for toxicity is unknown. We electroporated wild-type (WT), truncated WT or MTSOD1 expression constructs into the chick embryo spinal cord. MTSOD1 and truncated WTSOD1 (as small as 36 amino acid residues in length) aggregated in the cytoplasm of cells and caused cell death. These results suggest that MTSOD1 and truncated WTSOD1 lead to neural cell death because of misfolding, and that SOD1 peptides, possibly as a result of proteolytic digestion of MTSOD, play a role in FALS pathogenesis. Electroporation of the chick embryo spinal cord is a useful system in which to investigate neurodegenerative diseases because it provides efficient delivery of genes into neural cells in situ within a living organism.

Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins.

The proteins known as serum amyloid A (SAA) play major, but relatively uncharacterized, roles in the acute phase response and are important components of the innate immune system of humans and probably all vertebrates. N-terminal fragments of the inducible isoforms, SAA1 and SAA2, are the major constituents of fibrils formed during secondary or reactive amyloidosis. Little is known about the structure of SAA beyond secondary structure analyses and circular dichroism spectroscopic data indicating significant alpha helix conformation. Analysis of the primary structure of human SAA indicates probable homology to the N-terminal domain of hemocyanins of arthropods and suggests that approximately 80% of the molecule may consist of a helical bundle with the remaining portion of the C-terminus potentially disordered. This model of SAA suggests that proposed binding sites for laminin, fibronectin, and calcium are segregated to one face of the molecule and that the heparin/heparan binding site is found in the putatively disordered region of the protein. It is possible that removal of the N-terminal 76 amino acid fragment by proteolytic cleavage found generates an unstable entity that undergoes a helix to beta strand transition analogous to the fibril process of A-beta and prion peptides.

Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the beta sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys117 within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His125, which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the beta sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone.

Motivation: Methods that focus on secondary structures, such as Position Specific Scoring Matrices and Hidden Markov Models, have proved useful for assigning proteins to families. However, for assigning proteins to an attribute class within a family these methods may introduce more free parameters than are needed. There are fewer members and there is less variability among sequences within a family. We describe a method for organizing proteins in a family that exhibits up to an order of magnitude reduction in the number of parameters. The basis is the log odds ratio commonly used to measure similarity. We adapt this to characterize the sequence dissimilarities that give rise to attribute differentiation. This leads to the definition of Class Attribute Substitution Matrices (CLASSUM), a dual of the BLOSUM. Results: The method was applied to classify sequences hierarchically in the lambda and kappa subgroups of the immunoglobulin superfamily. Positions conferring class were identified based on the degree of amino acid variability at a position. The CLASSUM computed for these positions classified better than 90% of test data correctly compared with 35–50% for BLOSUM-62. The expected value for a random matrix is 14%. The results suggest that family-specific data-derived substitution matrices can improve the resolution of automated methods that use generic substitution matrices for searching for and classifying proteins.

Although more than 20 different proteins are now associated with the amyloidoses, the fibrils share many properties. Despite disparity in primary and tertiary structures of the subunit proteins, assembled fibrils exhibit similar morphology, binding of Congo red, interaction with Thioflavine T, formation of complexes with serum amyloid P component, apolipoprotein E, several glycosaminoglycans, the receptor for advanced glycation endproducts and cross-recognition by some monoclonal antibodies. Thus, it is probable that the mechanism of amyloid generation involves a generic process that can be evoked by most, if not all, proteins under conditions that degrade the native conformation. As suggested by others, the beta-helix or beta-roll conformation may be the unifying element of fibril conformations. Several proteins that have evolved to form physiologically useful amyloid-like fibrils, as well as some proteins associated with pathological amyloidoses, exhibit sequence repeat patterns that may facilitate beta-roll or beta-helix formation. Threading analyses of 2 natural amyloid-forming proteins, curli and human Pmel 17, indicate compatibility of their primary structures with both beta sandwich and beta-helix conformations, suggesting a possible innate conformational pliability. In addition, these results may suggest that the misfolded form of some proteins that are associated with conformational disease may be the native conformation of other proteins to which they are linked by evolution. Finally, since many matrix and structural proteins are known to incorporate numerous tandem repeat sequence elements, we propose that the mechanism of fibril formation is fundamentally related to a general protein assembly process that is integral to the generation of cells and tissues.

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.

We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.

Deposition of monoclonal immunoglobulin light chain (LC) aggregates in tissues is the hallmark of a class of fatal diseases with no effective treatment. In the most prevalent diseases two different types of LC aggregates are observed: fibrillar deposits in LC amyloidosis (AL) and granular aggregates in LC deposition disease (LCDD). The mechanisms by which a given LC forms either type of aggregate are not understood. Although some LCs are more aggregation-prone than others, this does not appear to be due to specific sequence determinants, but more likely from global properties that can be introduced by multiple somatic mutations. Moreover, a single LC isotype can sometimes form both fibrillar and granular aggregates within the same patient. To better understand how the different aggregation pathways arise, we developed a series of in vitro assays to analyze the formation of distinct aggregate types. The recombinant kappa IV LC (SMA) assembles into fibrils when agitated. We now show that SMA can also form granular aggregates upon exposure to copper, and that this aggregation can occur not only in vitro, but also in cells. A constellation of somatic mutations, consisting of His89/His94/Gln96, is sufficient to confer sensitivity to copper on wild-type kappa IV proteins. The formation of both types of aggregates is inhibited by synthetic peptides derived from the LC variable domain. However, the peptide that inhibits fibrillar aggregation is different from the peptide that inhibits copper-induced aggregation. Thus, distinct molecular surfaces of the LC underly each type of aggregate. We conclude that both the intrinsic properties of the sequence and extrinsic conditions govern the aggregation pathway of a LC.

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The E m is −285 mV when cysteines are at positions 121 and 305 and −295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects k cat but not K m values. In the single V121C and N122C mutants, the Cys in helix-5 affects the K m for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.

In systemic AL amyloidosis, fibrils are derived from a monoclonal immunoglobulin light chain produced by a plasma cell clone in the bone marrow.[1][1] AL amyloidosis may be associated with multiple myeloma but more commonly the plasma cell clone is not malignant. A monoclonal gammopathy of

Light chain deposition disease (LCDD) and light chain amyloidosis (AL) are disorders of monoclonal immunoglobulin deposition in which normally soluble serum precursors form insoluble deposits in tissues. A common feature in both is the clonal proliferation of B-cells that produce pathogenic light chains. However, the deposits in LCDD differ from those in AL in that they are ultrastructurally granular rather than fibrillar and do not bind Congo red or colocalize with amyloid P component or apolipoprotein E. The reason(s) for their differences are unknown but are likely multifactorial and related to their protein conformation and their interaction with other molecules and tissue factors in the microenvironment. Knowledge of the primary structure of the light chains in LCDD is very limited. In the present study two new κ 1 light chains from patients with LCDD are described and compared to seven other reported κ-LCDD proteins. The N-terminal amino acid sequences of light chain GLA extracted from the renal biopsy and light chain CHO from myocardial tissue were each identical to the respective light chains isolated from the urines and to the V-region amino acid sequences translated from the cloned cDNAs obtained from bone marrow cells. The germline V-region sequences, determined from the genomic DNA in both and in MCM, a previously reported κ 1 LCDD light chain, were identical and related to the L12a germline gene. The expressed light chains in all three exhibit amino acid substitutions that arise from somatic mutation and result in increased hydrophobicity with the potential for protein destabilization and disordered conformation.

Dysfunctional immunoglobulins (Igs) that are prone to aggregation are unavoidably generated by the diverse repertoire of B cells. Here, Fred Stevens and Yair Argon analyse the patterns of mutations that lead to pathological Igs, account for non-random mutations in human Ig sequences and suggest the exertion of selective forces, which contribute to determining and limiting the Ig repertoire.

Certain types of human light chains have the propensity to deposit pathologically as amyloid fibrils as evidenced by the preferential association of monoclonal lambda 6 proteins with AL amyloidosis. However, the molecular features that render such proteins amyloidogenic have not been elucidated. Based upon the demonstrated relationship between the thermodynamic stability of light chains and their propensity to aggregate in vitro, we have initiated studies where the thermodynamic properties and fibrillogenic potential of two recombinant (r) V lambda 6 molecules were compared. The first protein was generated from cDNA cloned from marrow-derived plasma cells from a patient (Wil) who had AL amyloidosis and renal amyloid deposits; the second was from a patient (Jto) with multiple myeloma in whom the lambda 6 protein was deposited not as amyloid but in the form of renal tubular casts. The thermodynamic stabilities of rV lambda 6Wil and -Jto were determined from chaotropic and thermal denaturation studies. Based upon the Delta GH2O, Delta H, Delta G25 degrees C, Tm, and Cm values, the rV lambda 6Wil was less stable than its nonamyloidogenic counterpart, rV lambda 6Jto. Measurement of fibril formation using a novel in vitro fibril forming assay demonstrated that although both rV lambda 6 proteins formed fibrils in vitro, Wil had a shorter lag time and exhibited faster kinetics under physiologic conditions. Comparative amino acid sequence analyses of these two components and other lambda 6 amyloid-associated light chains revealed that the Jto protein had certain primary structural features that we posit contributed to its increased stability and thus rendered this protein nonamyloidogenic. Our studies provide the first evidence that stabilizing interactions within the V L domain can influence the kinetics of light chain fibrillogenicity.

Immunoglobulin heavy chain-binding protein (BiP) is a member of the hsp70 family of chaperones and one of the most abundant proteins in the ER lumen. It is known to interact transiently with many nascent proteins as they enter the ER and more stably with protein subunits produced in stoichiometric excess or with mutant proteins. However, there also exists a large number of secretory pathway proteins that do not apparently interact with BiP. To begin to understand what controls the likelihood that a nascent protein entering the ER will associate with BiP, we have examined the in vivo folding of a murine λI immunoglobulin (Ig) light chain (LC). This LC is composed of two Ig domains that can fold independent of the other and that each possess multiple potential BiP-binding sequences. To detect BiP binding to the LC during folding, we used BiP ATPase mutants, which bind irreversibly to proteins, as “kinetic traps.” Although both the wild-type and mutant BiP clearly associated with the unoxidized variable region domain, we were unable to detect binding of either BiP protein to the constant region domain. A combination of in vivo and in vitro folding studies revealed that the constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond. Thus, the simple presence of a BiP-binding site on a nascent chain does not ensure that BiP will bind and play a role in its folding. Instead, it appears that the rate and stability of protein folding determines whether or not a particular site is recognized, with BiP preferentially binding to proteins that fold slowly or somewhat unstably.

AL amyloidosis is a disease process characterized by the pathologic deposition of monoclonal light chains in tissue. To date, only limited information has been obtained on the molecular features that render such light chains amyloidogenic. Although protein products of the major human VK and Vλgene families have been identified in AL deposits, one particular subgroup-λ6 - has been found to be preferentially associated with this disease. Notably, the variable region of λ6 proteins (V λ6) has distinctive primary structural features including the presence in the third framework region (FR3) of two additional amino acid residues that distinguish members of this subgroup from other types of light chains. However, the structural consequences of these alterations have not been elucidated. To determine if λ6 proteins possess unique tertiary structural features, as compared to light chains of other Vλ subgroups, we have obtained x-ray diffraction data on crystals prepared from two recombinant Vλ6 molecules. These c...

Background: The self-assembly properties of β domains are important features of diverse classes of proteins that include cell-adhesion molecules, surface receptors and the immunoglobulin superfamily. Immunoglobulin light-chain variable domains are well suited to the study of structural factors that determine dimerization, including how residues at the interface influence the preferred dimer arrangement. Results: Single-site mutants of light-chain variable domain Len, designated LenQ38E and LenK30T, formed ‘flipped' dimers in which one domain was rotated by about 180° compared with the native protein. The dimer in the native protein is similar to that found between variable domains in Fab immunoglobulin fragments. When compared to the native dimer, more surface area is buried, and more hydrogen bonds and salt bridges are formed between the monomers in the flipped conformation. Conclusions: Immunoglobulin light-chain variable domains can form a minimum of two distinct quaternary structures. Single-site mutations resulting from changes of one base, such as the exchange of Gln38 to Glu or Lys30 to Thr, change the ‘conventional' dimer of protein Len to a flipped arrangement. Native Len is not found in the flipped-domain dimer conformation because it would have excess positive electrostatic potential at the dimer interface that is not compensated by other forces. Excess negative or positive electrostatic potential at the dimer interface can have a determining effect on the mode of dimerization.

3 To whom correspondence should be addressed The formation of the antibody variable domain binding unit (Fv) is the net result of three competing assembly reactions. The affinities of concurrent homologous interactions of heavy and light chain variable domains limits the heterologous interaction leading to productive formation of the Fv. To address the possible role of light chain dimerization in this phenomenon, the Gln38 residue at the dimer interface of an immunoglobulin light chain variable domain (VL) was replaced by charged amino acids. The effects of these mutations on VL homodimer formation were monitored by small-zone size exclusion HPLC and the affinities of interaction were determined by computer simulation. Reduced VL homodimerization was observed in three of the four mutants, Q38R, Q38D and Q38K. The association constants for the Q38R and Q38D homodimers were 1.2310 4 and 3.2310 3 M ‐1 , respectively. This corresponded to a 20‐75-fold reduction in the homodimer association constant relative to the wild-type V L, which had an association constant of 2.4310 5 M ‐1 . Surprisingly, the fourth charge mutant, Q38E, had a higher association constant than the wild-type VL. The potential for charged residues to facilitate heterodimeric assembly of immunoglobulin domains was also tested. Heterodimerization was observed between the Q38D and Q38R VLs, but with an association constant of 4.7310 4 M ‐1 , approximately fivefold lower than that obtained for homodimerization of the native VL. In addition, replacement of the neutral, solventaccessible Gln38 residue with either Asp or Arg was found to be significantly destabilizing. These results suggest that charged residues could be introduced at immunoglobulin domain interfaces to guide heterodimer formation and to minimize unfavorable competing homologous associations. Nonetheless, these apparently simple modifications may also result in unintended consequences that are likely to depend upon structural features of particular variable domains.

The simulated structures of the enolases of Arabidopsis and the common ice plant contain a pair of Cys residues in the correct orientation to form a disulphide bond. Formation of this disulphide might be expected to affect the positioning of several residues in the active site. The enzyme in crude extracts of these two plants is activated by oxidation. Apparently formation of the disulphide crosslink enhances catalysis. The enolases from tomato leaves, maize roots and castor bean embryos lack one of these Cys residues and are not redox sensitive. It seems possible that enolase is redox-regulated by a cytosolic thioredoxin system in a limited number of plant species including ice plant and Arabidopsis.

Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. Tertiary structure modeling suggests that the redox-sensitivity of the chloroplast malate dehydrogenase (EC 1.1.1.82) is due to disulfide crosslinking of the carbon substrate and nucleotide-binding domains. Consistent with this suggestion, introduction of Cys residues in opposition to one another on the two domains of the Escherichia coli enzyme results in redox-sensitivity [Muslin EH et al. (1995) Biophys J 68: 2218-2223]. We have now substituted Cys residues into the bacterial malate dehydrogenase (EC 1.1.1.37) in positions that correspond more exactly to those postulated to be responsible for the redox-sensitivity of the chloroplast enzyme. The introduction of one pair of Cys residues renders the enzyme redox-sensitive, but the introduction of the alternate pair does not. Energy minimization calculations suggest that the difference in redox-sensitivity is consistent with differences in the energy required for formation of the disulfide bond.

Antibody light chains of the kappa subgroup are the predominant light chain component in human immune responses and are used almost exclusively in the antibody repertoire of mice. Human kappa light chains comprise four subgroups. To date, all crystallographic studies of human kappa light chains were carried out on proteins of the kappaI subgroup. The light chain produced by multiple myeloma patient Len. was of the kappaIV subgroup, it differed by only one residue from the germ-line gene encoded protein. The variable domain fragment of the light chain was crystallized from ammonium sulfate in space group C222(1). The crystal structure was determined by molecular replacement and refined at 1.95 A resolution to an R-factor of 0.15. Protein Len has six additional residues in its CDR1 segment compared to the kappaI proteins previously characterized. The kappaIV variable domain, Len, differs in only 23 of 113 residues from murine kappa light chain McPC603. The RMS deviation upon superimposing their alpha-carbons was 0.69 A. The CDR1 segment of the human and murine variable domains have the same length and conformation although their amino acid sequences differ in 5 out of 17 residues. Structural features were identified that could account for the significantly higher stability of the human kappaIV protein relative to its murine counterpart. This human kappaIV light chain structure is the closest human homolog to a murine light chain and can be expected to facilitate detailed structural comparisons necessary for effective humanization of murine antibodies.

Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested by both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddlelike surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.

Resume L'un des problemes non encore parfaitement resolus de la biochimie des plantes est l'identification des residus cysteines qui interviennent dans la regulation des enzymes chloroplastiques activees et desactivees par la lumiere. La modelisation par homologie basee sur la connaissance des sequences de ces enzymes et des structures tridimensionnelles des enzymes homologues a maintenant permis une approche de l'identification des residus Cys sensibles au potentiel redox pour quatre enzymes chloroplastiques activees par la lumiere. Dans chaque cas les disulfites regulateurs ne sont pas situes dans le site actif, mais plutot dans des sites qui affectent la flexibilite ou la conformation de l'enzyme, modifiant ainsi ses caracteres catalytiques. Chez la glyceraldehyde-3-P deshydrogenase et la malate deshydrogenase, les mouvements inter-domaines seraient ainsi limites. Pour la fructose bisphosphatase et la sedoheptulose bisphosphatase, les residus Cys regulateurs sont localises sur le domaine lie au nucleotide dans une region connue pour sa sensibilite a l'AMP, effecteur allosterique des autres fructose bisphosphatases. Les resultats des experiences de mutagenese dirigee faites jusqu'a ce jour sont generalement en accord avec l'hypothese du blocage des domaines. La sensibilite au potentiel redox d'un certain nombre d'enzymes cytosoliques suggere que la modulation reductrice pourrait avoir lieu hors des chloroplastes dans les feuilles, et aussi dans les racines, les tiges et les graines en germination chez les plantes vertes. Cette meilleure comprehension du mecanisme de regulation par le potentiel redox devrait permettre de nouvelles approches du mecanisme de la regulation des activites enzymatiques et conduire a des applications en biotechnologie.

Light chain deposition disease is a severe complication of certain immunoproliferative disorders, due to the secretion of a monoclonal light chain which precipitates close to basement membranes of several tissues. A kappa isotype restriction and an unusual frequency of a variable region subgroup (VkappaIV) suggest that precise structural features govern the propensity of pathogenic light chains to precipitate in extracellular spaces. We studied primary structures of light chains from six patients with light chain deposition disease in comparison with light chains from other pathological conditions. Sequence alignment revealed the presence of certain amino acids only in light chain deposition disease, in particular non-polar replacing hydrophilic residues. To determine the role of these residues, structures of the variable domain from four kappa chains belonging to VkappaI and VkappaIV subgroups responsible for deposition disease were modeled using known immunoglobulins as templates. The most evident structural features shared by all pathogenic light chains were hydrophobic residues exposed to the solvent in complementarity determining regions 1 or 3. In contrast to immunoglobulin light chain-related amyloidosis, where deposition of organized material might be due to electrostatic interactions between light chain dimers, hydrophobic interactions could enhance amorphous precipitation in non-amyloid light chain deposition disease.

The deduced amino acid sequences of the Brassica napus and sugar cane ( Saccharum sp.) cytosolic fructose bisphosphatases (EC 3.1.3.11) have appeared recently. When the three-dimensional structure of the Brassica napus bisphosphatase was modeled residue 92, previously identified as a potential redox-sensitive regulatory cysteine in the cytosolic enzymes from potato, sugarbeet and spinach [L.E. Anderson, et al., Planta 196 (1995) 118–124], was a serine. (Numbering according to Protein Data Bank entry 4FBP.) Instead there is a Cys at position 110, close enough to Cys-114, the second member of the potential regulatory cysteine pair in the other cytosolic fructose bisphosphatases, to suggest the possibility of disulfide bond formation and the enzyme is redox-sensitive. The sugar cane enzyme, like the other three cytosolic fructose bisphosphatases, contains Cys-92 and Cys-114. It also is redox-sensitive. Apparently a disulfide anywhere in the region of Cys-114, -92 and -110 can function in the redox-modulation of the activity of this enzyme.

The chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) of the green alga Chlamydomonas reinhardtii is reductively light activated. Homology modeling indicates that the only potential disulfide-forming cysteine residues in this enzyme are the same cysteine residues suggested to be responsible for redox-sensitivity of the higher plant enzyme (Li D, Stevens FJ, Schiffer M and Anderson LE (1994) Biophys J 67: 29–35). Apparently, the three additional cysteines in the higher plant enzyme are not necessary for light activation. The putative regulatory cysteines are juxtaposed across the domain interface and when oxidized will crosslink the domains. This would be expected to interfere with interdomain movement and catalysis. This is the first report of reductive light activation of this enzyme in a green alga.

The mitochondrial citrate synthases (EC 4.1.3.7) of pummelo, potato and Arabidopsis are activated in crude extracts by dithiothreitol treatment and/or inactivated by the strong oxidizing agent diamide. Surprisingly, homology modeling reveals a potential disulfide involving two cysteine residues which are also present in the redox-insensitive model enzyme, pig heart citrate synthase. Energy minimization calculations suggest that differences in the charge distribution enhance disulfide bond formation in the plant mitochondrial citrate synthase and inhibit disulfide bond formation in the mammalian enzyme.

In the stimulated three-dimensional structure of the Chlamydomonas reinhardtii sedoheptulose bisphosphatase (EC 3.1.3.37) there are two cysteine residues close enough to one another to form a redox-sensitive disulfide bond which would cross-link the nucleotide and carbon substrate domains. Examination of the redox modulation of this sedoheptulose bisphosphatase confirms that it resembles the higher plant enzyme in being activated by reduction. In the wheat and Arabidopsis enzymes, for which there is sequence information and which, like the Chlamydomonas enzyme, can be modeled, both redox-sensitive Cys residues appear to be located on the regulatory nucleotide-binding domain. Apparently different Cys residues are involved in modulation in the algal and higher plant sedoheptulose bisphosphatases.

The formation and pathological deposition of amyloid fibrils are defining features of many acquired and inherited disorders, including primary or light-chain-associated amyloidosis, Alzheimer`s disease, and adult-onset diabetes. No pharmacological methods exist to block this process or to effect the removal of fibrils from tissue, and thus, little can be done to prevent organ failure and ultimate death that result from deposition of amyloid. Knowledge of the pathogenesis, treatment, or prevention of these presently incurable diseases is limited due to the relative paucity of information regarding the biophysical basis of amyloid formation. Antibody light chains of different amino acid sequence show differential amyloid-forming tendencies and, as such, can provide insight into the structural organization of amyloid fibrils as well as into basic mechanisms of protein self-assembly. We have compared primary structures of 180 human monoclonal light chains and have identified particular residues and positions within the variable domain that differentiate amyloid-from nonamyloid-associated proteins. We propose a molecular model that accounts for amyloid formation by antibody light chains and might also have implications for other forms of amyloidosis. 24 refs., 2 figs., 1 tab.

Monoclonal human light chains, i.e. Bence Jones proteins, and their recombinant variable fragments (VL) were screened for proteolytic activity using peptide-methylcoumarinamide (peptide-MCA) conjugates and vasoactive intestinal polypeptide (VIP) as substrates. Sixteen of 21 Bence Jones proteins and one of three VL fragments were capable of detectable cleavage of one or more substrates. The magnitude and kinetic characteristics of the activity varied with different substrates. Among the peptide-MCA substrates, the presence of tripeptide or tetrapeptide moieties with a basic residue at the scissile bond generally favored expression of the activity. The influence of N-terminal flanking residue recognition was evident from differing values of Km and kcat (turnover number) observed using different Arg-containing peptide-MCA substrates. Different light chains displayed different kinetic parameters for the same substrate, suggesting unique catalytic sites. Hydrolysis of VIP was characterized by nanomolar Michaelis-Menten constants (Km), suggesting comparatively high affinity recognition of this peptide. The 25-kDa monomer and the 50-kDa dimer forms of one light chain preparation were resolved by gel filtration in 6 M guanidine hydrochloride. Following renaturation, the monomer displayed 51-fold greater peptide-MCA-hydrolyzing activity than the dimer. A renatured VL domain prepared by gel filtration in 6 M guanidine hydrochloride displayed VIP-hydrolyzing activity in the 12.5-kDa peak fractions. These results provide evidence for the proteolytic activity of certain human light chains and imply that this phenomenon may have a pathophysiological significance.

One of the cysteine residues believed to be necessary for reductive light activation is lacking in the only red algal NADP-linked glyceraldehyde-3-P dehydrogenases for which sequences are available, namely Gracilaria verrucosa (Hudson) Papenfuss and Chondrus crispus Stackhouse. Consistent with the mechanism of light modulation proposed for this enzyme, which involves reduction of domain movement-restricting disulfide bonds, it is not reductively activated in Chondrus crispus extracts, and it is not light-activated in whole cells or dithiothreitol (DTT) activated in extracts of the North American species Gracilaria tikvahiae McLachlan. Fructosebisphosphatase and glucose-6-P dehydrogenase, two enzymes for which sequence information from these algae is not yet available, are both activated in crude extracts by DTT treatment, but only fructosebisphosphatase is light-activated in intact Gracilaria.

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29-35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.

The sequence and structure of a second human kappa 1 immunoglobulin light-chain variable domain, Wat, has been determined. The R-factor is 15.7% for 1.9-A data. One hundred and ninety-five water molecules were identified; 30 water molecules were located in identical positions in each of the monomers. Some of the water molecules are integral parts of the domains. This light chain is encoded by the same variable domain gene that encoded the previously characterized kappa I variable domain, Rei. Due to limited somatic mutation, the two highly homologous proteins differ in only 20 of the 108 residues. Wat crystallized in space group P6(4) while Rei crystallized in space group P6(1); in both crystals, the asymmetric unit was the noncovalent dimer. Although the basic domain structure is the same for both proteins, the relative positions of the domains within the two dimers differ. This difference is most likely accounted for by the replacement of Tyr36 in Rei by Phe in the Wat protein. Residue Tyr36 is part of the hydrogen-bonding network in the interface between the domains in Rei. Losing the hydrogen-bonding capability of residue 36 by replacement of Tyr by Phe alters the network of hydrogen bonds between the domains, resulting in a different domain-domain contact. The details of lattice contacts in the two crystals were compared. One type of contact that extends the beta-sheet of the individual domains was conserved, but because it involved different symmetry elements within the crystal, different crystal packing resulted. In the Wat crystal, one of the contacts shows an example of how a symmetrical binding site can "bind" an asymmetrical object. Further, the examination of the Wat crystal also illustrates how the different crystalline environments of the domains of the dimer results in different distributions of temperature factors for the residues within the domains.