The solution structure and stability of N-terminally truncated b2-microglobulin~DN6b2-m!, the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that DN6b2-m has a free energy of stabilization that is reduced by 2.5 kcal0mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at mM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that DN6b2-m is significantly less protected than its wild-type counterpart. Analysis of DN6b2-m by NMR shows that this loss of protection occurs in b strands I, III, and part of II. At mM concentration gel filtration analysis shows that DN6b2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of b2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that DN6b2-m could be a key intermediate of a proteolytic pathway of b2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of b2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between b-strands far removed from this constrain is greatly perturbed.Keywords: amyloidosis; b2-microglobulin; hydrogen exchange mass spectrometry; limited proteolysis; NMR; protein folding Amyloidoses are diseases caused by tissue deposition of protein aggregate organized in an ordered b-sheet structure. The conversion of globular proteins to insoluble fibrillar aggregates requires significant conformational changes, such as the loss of tertiary and quaternary interactions or conversion of a to b secondary structurẽ Sunde & Blake, 1998!. Of the 17 or so proteins implicated in amyloidoses the fibril morphology is indistinguishable and there does not appear to be any common features that link the soluble precursor proteins. For many of these proteins, the amyloid fibril formation is facilitated by amino acid mutations that destabilize the native state and confer a structural flexibility to the molecule, but other proteins like IAPP, wild-type TTR, and b2-microglobulin

The solution structure of human ␤2-microglobulin (␤2-m), the nonpolymorphic component of class I major histocompatibility complex (MHC-I), was determined by 1 H NMR spectroscopy and restrained modeling calculations. Compared to previous structural data obtained from the NMR secondary structure of the isolated protein and the crystal structure of MHC-I, in which the protein is associated to the heavy-chain component, several differences are observed. The most important rearrangements were observed for (1) strands V and VI (loss of the C-terminal and N-terminal end, respectively), (2) interstrand loop V-VI, and (3) strand I, including the N-terminal segment (displacement outward of the molecular core). These modifications can be considered as the prodromes of the amyloid transition. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches at the interface with heavy chain and the nearby region at the surface charge cluster of the C-terminal segment. As a result, the molecule is placed in a state in which even minor charge and solvation changes in response to pH or ionic-strength variations can easily compromise the hydrophobic/hydrophilic balance and trigger the transition into a partially unfolded intermediate that starts with unpairing of strand I and leads to polymerization and precipitation into fibrils or amorphous aggregates. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu 2+ binding, which is shown to occur primarily at His 31 and involve partially also His 13, the next available His residue along the partial unfolding pathway.Keywords: ␤2-microglobulin; amyloid; dialysis-related amyloidosis; protein structure; NMR of proteins; unfolding Supplemental material: See www.proteinscience.org. (␤2-m) is the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I). It consists of 99 residues with a single disulfide bridge between the two Cys residues of the sequence at positions 25 and 80 and folds into the classical ␤-sandwich motif of the immunoglobulin superfamily, as shown by the crystal structure of MHC-I (Bjorkman et al. 1987). The systemic deposition of ␤2-m fibrils is associated to dialysis-related amyloidosis (DRA; Gejyo et al. 1985), a disease which arises in individuals with chronic renal failure as the inescapable complication of long-term hemodialysis. More than 90% of Reprint requests to: Prof. G. Esposito, Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, P.le Kolbe 4, 33100 Udine, Italy; e-mail: gesposito@mail.dstb.uniud.it; fax: 39-0432-494301. ␤2-microglobulinAbbreviations: ␤2-m, ␤2-microglobulin; ⌬N6␤2-m, ␤2-microglobulin 7-99 fragment; 1D, 2D, 3D, one, two, three dimension; DQF COSY, double quantum filtered correlation spectroscopy; DRA, dialysis related amyloidosis; GdnHCl, guanidinium chloride; HLA, human lukocyte antigen; MHC-I, class I major histocompatibility complex; NOE, nuclear Overhauser effect; NOESY, nuclear O...

Three variants of human ␤ 2 -microglobulin (␤ 2 -m) were compared with wild-type protein. For two variants, namely the mutant R3A␤ 2 -m and the form devoid of the N-terminal tripeptide (⌬N3␤ 2 -m), a reduced unfolding free energy was measured compared with wild-type ␤ 2 -m, whereas an increased stability was observed for the mutant H31Y␤ 2 -m. The solution structure could be determined by 1 H NMR spectroscopy and restrained modeling only for R3A␤ 2 -m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Y␤ 2 -m and ⌬N3␤ 2 -m. Precipitation and unfolding were observed over time periods shorter than 4 -6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for ␤ 2 -m fibrillogenesis. The mechanism is consistent with the previous and present results on ␤ 2 -m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.Over the last several years, an overwhelming number of reports have addressed the phenomenon of amyloidogenesis. The interest in the subject stems not only from the social relevance of amyloid pathologies such as Alzheimer's disease, or spongiform encephalopathy, or the various systemic amyloidoses, but also from the general implications in the issue of protein folding.Amyloidoses have been recognized as conformational diseases that arise from the conversion of globular proteins into insoluble fibrillar aggregates (1). Despite the diversity of the involved proteins, amyloid fibrils exhibit a common structure known as cross-␤ structure, which appears to be a particularly stable, generic protein fold, accessible to many polypeptide chains under specific conditions in vitro and in vivo (2). The amyloid deposition of ␤ 2 -microglobulin (␤ 2 -m), 1 the nonpolymorphic light chain of the class I major histocompatibility complex (MHC-I), is associated to dialysis-related amyloidosis (3). The disease is the result of long term hemodialysis in individuals with chronic renal failure, a widespread pathology with high social costs that are further increased by the inevitable dialysis-related amyloidosis complication. Recently ankylosing spondylitis has also been proposed to originate from ␤ 2 -m deposition (4). We determined the solution structure of isolated ␤ 2 -m by NMR spectroscopy (5) and showed that the most important rearrangements of the protein, with respect to its structure in MHC-I, were observed for strands D and E, interstrand loop D-E, and strand A, including the N-terminal segment. We stated that these modifications can be conside...

The extracellular matrix protein EMILIN1 (elastin microfibril interface located protein 1) is implicated in maintaining blood pressure homeostasis via the N-terminal elastin microfibril interface domain and in trophoblast invasion of the uterine wall via the globular C1q (gC1q) domain. Here, we describe the first NMR-based homology model structure of the human 52-kDa homotrimer of the EMILIN1 gC1q domain. In contrast to all of the gC1q (crystal) structures solved to date, the 10-stranded ␤-sandwich fold of the gC1q domain is reduced to nine ␤ strands with a consequent increase in the size of the central cavity lumen. An unstructured loop, resulting from an insertion unique to EMILIN1 and EMILIN2 family members and located at the trimer apex upstream of the missing strand, specifically engages the ␣4␤1 integrin. Using both Jurkat T and EA.hy926 endothelial cells as well as site-directed mutagenesis, we demonstrate that the ability of ␣4␤1 integrins to recognize the trimeric EMILIN1 gC1q domain mainly depends on a single glutamic acid residue (Glu 933 ). Static and flow adhesion of T cells and haptotactic migration of endothelial cells on gC1q is fully dependent on this residue. Thus, EMILIN1 gC1q-␣4␤1 represents a unique ligand/receptor system, with a requirement for a 3-fold arrangement of the interaction site.EMILIN1 (elastin microfibril interface located protein 1) is a secreted extracellular matrix multidomain glycoprotein (1, 2). It is characterized by a unique arrangement of structural domains, including the elastin microfibril interface domain at the N terminus, an ␣-helical domain predicted to form a coiledcoil structure in the central part of the molecule, a short collagenous sequence, and a region homologous to the globular domain of C1q (gC1q domain) 4 at the C-terminal end (3, 4). Although the role of the coiled-coil region has not yet been elucidated, it has conclusively been demonstrated that EMILIN1 interacts with pro-tumor growth factor-␤ (5) through the elastin microfibril interface domain (6). EMILIN1 deficiency causes systemic arterial hypertension, and the expression of EMILIN1 at physiological levels by binding to pro-tumor growth factor-␤ prevents its maturation by protein convertases (5). Thus, EMILIN1 favorably located at the subendothelium of blood vessels is a new specific antagonist of tumor growth factor-␤, and the function of this constituent of elastic tissues is linked to the pathogenesis of hypertension. The C-terminal gC1q domain is involved in the oligomerization of EMILIN1 (7), in cell adhesion and migration via interaction with the ␣4␤1 integrin (8), and in trophoblast invasion (9).The gC1q signature is found in a variety of proteins, and the essential features of the specific structure-function relationship were recognized with the elucidation of the crystal structure of the homotrimeric gC1q domain of mouse ACRP30 (adipocyte complement-related protein of 30 kDa) (10). It suggested a structural and evolutionary link between the tumor necrosis factor and the gC1q domains and le...

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EMILIN1 is a glycoprotein of elastic tissues that has been recently linked to the pathogenesis of hypertension. The protein is formed by different independently folded structural domains whose role has been partially elucidated. In this paper the solution structure, inferred from NMR-based homology modelling of the C-terminal trimeric globular C1q domain (gC1q) of EMILIN1, is reported. The high molecular weight and the homotrimeric structure of the protein required the combined use of highly deuterated (15)N, (13)C-labelled samples and TROSY experiments. Starting from a homology model, the protein structure was refined using heteronuclear residual dipolar couplings, chemical shift patterns, NOEs and H-exchange data. Analysis of the gC1q domain structure of EMILIN1 shows that each protomer of the trimer adopts a nine-stranded beta sandwich folding topology which is related to the conformation observed for other proteins of the family. Distinguishing features, however, include a missing edge-strand and an unstructured 19-residue loop. Although the current data do not allow this loop to be precisely defined, the available evidence is consistent with a flexible segment that protrudes from each subunit of the globular trimeric assembly and plays a key role in inter-molecular interactions between the EMILIN1 gC1q homotrimer and its integrin receptor alpha4beta1.

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