Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis.

Although both light chain amyloidosis (AL) and deposition disease (LCDD) involve the aggregation of light chain V(L) domains into highly insoluble deposits, the factors which determine both disease onset and type (amyloid fibrils (AL) or granular deposits (LCDD)) are not clear. Previously, we showed that the AL-associated replacement Arg61 --> Asn, introduced as a point mutation into the kappa V(L) domain REI, greatly destabilizes the domain and renders it susceptible to the formation of ordered, fibril-like aggregates in vitro. The importance of Arg61 for stability may be due to the role of this residue in making a key, conserved salt bridge with Asp82 located on an adjacent loop. Here we show that an Asp82 --> Ile replacement, recently identified in a V(L) associated with LCDD, also highly destabilizes REI as a point mutation and makes it susceptible to in vitro aggregate formation. The D82I aggregate, however, is dramatically different in morphology from aggregates obtained from amyloid-associated mutants, suggesting that specific amino acid residue changes can control not only the onset of aggregation disease but also aggregate morphology and disease type.

Mutational effects on inclusion body formation in the periplasmic expression of the immunoglobulin VL domain REI.

BACKGROUND: Inclusion body (IB) formation in bacteria is an important example of protein misassembly, a phenomenon which also includes folding-dependent aggregation in vitro and amyloid deposition in human disease. Previous studies of mutational effects in other systems implicate the stability of a folding intermediate-rather than the native state-as playing a key role in IB formation. To contribute to an understanding of the comparative biophysics of VL misassembly in different biological settings, we have studied mutation-dependent periplasmic IB formation by the VL domain REI in Escherichia coli. RESULTS: A series of mutants were produced in periplasmic IBs, where, in all cases, the signal peptide was removed. In addition, the intradomain disulfide was clearly formed before deposition into IBs. IB formation in these mutants does not correlate with monomer/dimer equilibrium constants, but does correlate with the thermodynamic stability of the native state. CONCLUSIONS: The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date. As equilibrium unfolding intermediates have also been implicated in light chain amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases.

A paradigm for drug discovery using a conformation from the crystal structure of a presentation scaffold.

We describe a structural validation of the use of presentation scaffolds for control and elucidation of bioactive conformations of peptides. The protein REI-RGD34--produced by inserting the sequence RIPRGDMP into the CDR1 loop region of the immunoglobulin VL domain REI--strongly inhibits fibrinogen binding to the integrins alpha IIb beta 3 and alpha V beta 3. In the X-ray crystal structure of their protein at 2.4 A resolution, the RGD-containing loop exhibits defined electron density that is consistent with models for the bioactive conformations of ligands of these receptors based on previous small-molecule studies. Furthermore, a search of a small-molecule database with conformational information derived from the structure of REI-RGD34 identified constrained peptides and peptidomimetics known to be antagonists of the platelet receptor alpha IIb beta 3.

Destabilizing loop swaps in the CDRs of an immunoglobulin VL domain.

It is generally believed that loop regions in globular proteins, and particularly hypervariable loops in immunoglobulins, can accommodate a wide variety of sequence changes without jeopardizing protein structure or stability. We show here, however, that novel sequences introduced within complementarity determining regions (CDRs) 1 and 3 of the immunoglobulin variable domain REI VL can significantly diminish the stability of the native state of this protein. Besides their implications for the general role of loops in the stability of globular proteins, these results suggest previously unrecognized stability constraints on the variability of CDRs that may impact efforts to engineer new and improved activities into antibodies.

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.

A role for destabilizing amino acid replacements in light-chain amyloidosis.

Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (VL) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain. In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret. In this paper we identify particular amino acid replacements at specific positions in the VL domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis. Analysis of the structural model for the VL domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability. Using an Escherichia coli expression system, we prepared single-point mutants of REI VL incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions. These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI VL remains soluble. Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding. The results are consistent with a mechanism for the disease process in which the VL domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state.

The primary structures of the flavodoxins from two strains of Desulfovibrio gigas. Cloning and nucleotide sequence of the structural genes.

The structural genes coding for the flavodoxin proteins from two different strains of the sulfate-reducing bacteria Desulfovibrio gigas (ATCC 19364/NCIB 9332 and ATCC 29494/DSM 496) have been identified, cloned and the nucleotide sequence established. The protein sequences derived from the gene from each strain share a sequence identity of 66% with regions directly involved in binding the flavin mononucleotide cofactor being the most homologous. Both aromatic residues that flank the flavin isoalloxazine ring in the crystal structure of the flavodoxin from D. vulgaris, i.e., Trp-60 and Tyr-98, are also present in these flavodoxin proteins. These observations stand in contrast to reports that the flavodoxin from Desulfovibrio gigas contains a single tryptophan residue which is located distant from the flavin binding site. Therefore, the FMN binding site of this flavodoxin is not distinct from the other Desulfovibrio flavodoxins in this regard.

Primary structure of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough): cloning and nucleotide sequence of the reductase gene.

The nucleotide sequence encoding the structural gene (651 bp) and flanking regions for the assimilatory-type sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) was determined after cloning a 1.4 kb HindIII/SalI genomic fragment possessing the gene into Bluescript pBS(+)KS. The primary structure of the protein was deduced, and the molecular mass of the apoprotein was estimated as 24 kDa. The amino acid sequence of the polypeptide shows some similarities at putative [Fe4S4] cluster binding sites in comparison with the heme protein subunit of the larger Escherichia coli and Salmonella typhimurium sulfite reductases and spinach nitrite reductase. This is the first reported sequence of a member of a new class of low molecular weight assimilatory sulfite-reducing enzymes recently identified in a number of anaerobic bacteria [Moura, I., Lina, A. R., Moura, J. J. G., Xavier, A. V., Fauque, G., Peck, H. D., & Le Gall, J. (1986) Biochem. Biophys. Res. Commun. 141, 1032-1041].

Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans.

The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of HindIII-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desulfovibrio vulgaris [Hildenborough] (Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). Ultimately, a 1.8 kb TaqI fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15,726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. vulgaris. Regions of the polypeptide which form the flavin mononucleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxazine ring in the D. vulgaris structure, i.e., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin.

Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens.

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.