Alessandra Corazza

The solution structure of human β2‐microglobulin reveals the prodromes of its amyloid transition

The solution structure of human β2‐microglobulin (β2‐m), the nonpolymorphic component of class I major histocompatibility complex (MHC‐I), was determined by 1H 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 Cu2+ 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.

Collagen Plays an Active Role in the Aggregation of β2-Microglobulin under Physiopathological Conditions of Dialysis-related Amyloidosis

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils ofβ2-microglobulin (β2-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, β2-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology ofβ2-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in β2-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role inβ2-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-l-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-l-lysine is dissolved in solution with β2-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as β2-m, that decay on moving away from the fiber surface.

The Controlling Roles of Trp60 and Trp95 in β2-Microglobulin Function, Folding and Amyloid Aggregation Properties

Amyloidosis associated to hemodialysis is caused by persistently high beta(2)-microglobulin (beta(2)m) serum levels. beta(2)m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no beta(2)m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to beta(2)m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of beta(2)m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60-->Gly mutation has a threefold effect: it stabilizes beta(2)m, inhibits beta(2)m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the beta(2)m core; the Trp95-->Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar beta(2)m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in beta(2)m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type beta(2)m towards self-aggregation into amyloid fibrils.

Effect of Tetracyclines on the Dynamics of Formation and Destructuration of β2-Microglobulin Amyloid Fibrils

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC50 is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.

β2-Microglobulin isoforms display an heterogeneous affinity for type I collagen

It has been claimed that β2‐microglobulin (β2‐m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the β2‐m amyloid deposits that target the osteo‐articular system. The binding parameters of the interaction between collagen and β2‐m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of β2‐m. Wild‐type β2‐m binds collagen type I with a Kd of 4.1 × 10−4 M and type II with 2.3 × 10−3 M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of β2‐m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of β2‐m truncated at the third N‐terminal residue does not differ from that reported for the wild‐type protein. Increased affinity for collagen type I is found in the case of N‐terminal truncated species lacking of six residues. The Kd of this species is 3.4 × 10 −5 M at pH 7.4 and its affinity increases to 4.9 × 10−6 M at pH 6.4. Fluctuations of the affinity caused by β2‐m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.

Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin ROLES OF SHEAR FLOW, HYDROPHOBIC SURFACES, AND α-CRYSTALLIN

Background: We recently discovered the first natural human β2-microglobulin variant, D76N, as an amyloidogenic protein. Results: Fluid flow on hydrophobic surfaces triggers its amyloid fibrillogenesis. The α-crystallin chaperone inhibits variant-mediated co-aggregation of wild type β2-microglobulin. Conclusion: These mechanisms likely reflect in vivo amyloidogenesis by globular proteins in general. Significance: Our results elucidate the molecular pathophysiology of amyloid deposition. Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by 1H‐NMR spectroscopy and X‐ray crystallography. Solution and crystal structures (1.27 Å resolution, R‐factor 13.7%) were obtained on the full‐length protein and on an N‐truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same βαββαβ topology previously described for other members of the AcP family from mesophilic sources. The unstructured N‐terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25–37°C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C‐terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8°C and an unfolding free energy, ΔG  U‐FH 2O , at 28°C and 81°C of 48.7 and 20.6 kJ mol−1, respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcPs display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcPs revealed the importance of a ion pair network stabilizing particularly the β‐sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher α‐helical propensity. Proteins 2006.

Probing the influence of citrate-capped gold nanoparticles on an amyloidogenic protein.

Nanoparticles (NPs) are known to exhibit distinct physical and chemical properties compared with the same materials in bulk form. NPs have been repeatedly reported to interact with proteins, and this interaction can be exploited to affect processes undergone by proteins, such as fibrillogenesis. Fibrillation is common to many proteins, and in living organisms, it causes tissue-specific or systemic amyloid diseases. The nature of NPs and their surface chemistry is crucial in assessing their affinity for proteins and their effects on them. Here we present the first detailed structural characterization and molecular mechanics model of the interaction between a fibrillogenic protein, β2-microglobulin, and a NP, 5 nm hydrophilic citrate-capped gold nanoparticles. NMR measurements and simulations at multiple levels (enhanced sampling molecular dynamics, Brownian dynamics, and Poisson-Boltzmann electrostatics) explain the origin of the observed protein perturbations mostly localized at the amino-terminal region. Experiments show that the protein-NP interaction is weak in the physiological-like, conditions and do not induce protein fibrillation. Simulations reproduce these findings and reveal instead the role of the citrate in destabilizing the lower pH protonated form of β2-microglobulin. The results offer possible strategies for controlling the desired effect of NPs on the conformational changes of the proteins, which have significant roles in the fibrillation process.

Bluues server

MOTIVATION Electrostatic calculations are an important tool for deciphering many functional mechanisms in proteins. Generalized Born (GB) models offer a fast and convenient computational approximation over other implicit solvent-based electrostatic models. Here we present a novel GB-based web server, using the program Bluues, to calculate numerous electrostatic features including pKa-values and surface potentials. The output is organized allowing both experts and beginners to rapidly sift the data. A novel feature of the Bluues server is that it explicitly allows to find electrostatic differences between wild-type and mutant structures. AVAILABILITY The Bluues server, examples and extensive help files are available for non-commercial use at URL: http://protein.bio.unipd.it/bluues/.