9 research outputs found
Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis
Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(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 beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(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
Solution structure of beta(2)-microglobulin and insights into fibrillogenesis
The solution structure of human b2-microglobulin (b2-m) was determined by 1H NMR spectroscopy and restrained modeling calculations.
Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain
component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand CV and loop DE,
shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes
in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same
mechanism accounts for the partial unfolding and fiber formation subsequent to Cu2+ binding which is shown to occur primarily at H31.
Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via
surface charge cluster. Mutants or truncated forms of h2-m can be designed to remove the instability from H31 titration or to enhance the
instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either
in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained
The solution structure of EMILIN1 globular C1q domain reveals a disordered insertion necessary for interaction with the alpha4beta1 integrin
The extracellular matrix protein EMILIN1 (elastin microfi-
bril interface located protein 1) is implicated in maintaining
blood pressure homeostasis via the N-terminal elastin microfi-
bril 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 \u2424-sandwich fold of the gC1q domain is reduced to
nine \u2424 strands with a consequent increase in the size of the cen-
tral cavity lumen. An unstructured loop, resulting from an inser-
tion unique to EMILIN1 and EMILIN2 family members and
located at the trimer apex upstream of the missing strand, spe-
cifically engages the \u24234\u24241 integrin. Using both Jurkat T and
EA.hy926 endothelial cells as well as site-directed mutagenesis,
we demonstrate that the ability of \u24234\u24241 integrins to recognize
the trimeric EMILIN1 gC1q domain mainly depends on a single
glutamic acid residue (Glu933). Static and flow adhesion of T
cells and haptotactic migration of endothelial cells on gC1q is
fully dependent on this residue. Thus, EMILIN1 gC1q-\u24234\u24241
represents a unique ligand/receptor system, with a requirement
for a 3-fold arrangement of the interaction site