Paolo Viglino

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.

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5′‐TAAT‐3′. The homeodomain of thyroid transcription factor 1 (TTF‐1HD) binds to sequences containing a 5′‐CAAG‐3′ core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF‐1HD by both structural and functional approaches. As already suggested by the three‐dimensional structure of TTF‐1HD, the DNA binding specificities of the TTF‐1, Antennapedia and Engrailed homeodomains, either wild‐type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3′ end of the core motif 5′‐NAAN‐3′. The nucleotide at the 5′ position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF‐1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Biomolecular Electrostatics with the Linearized Poisson-Boltzmann Equation

Electrostatics plays a key role in many biological processes. The Poisson-Boltzmann equation (PBE) and its linearized form (LPBE) allow prediction of electrostatic effects for biomolecular systems. The discrepancies between the solutions of the PBE and those of the LPBE are well known for systems with a simple geometry, but much less for biomolecular systems. Results for high charge density systems show that there are limitations to the applicability of the LPBE at low ionic strength and, to a lesser extent, at higher ionic strength. For systems with a simple geometry, the onset of nonlinear effects has been shown to be governed by the ratio of the electric field over the Debye screening constant. This ratio is used in the present work to correct the LPBE results to reproduce fairly accurately those obtained from the PBE for systems with a simple geometry. Since the correction does not involve any geometrical parameter, it can be easily applied to real biomolecular systems. The error on the potential for the LPBE (compared to the PBE) spans few kT/q for the systems studied here and is greatly reduced by the correction. This allows for a more accurate evaluation of the electrostatic free energy of the systems.

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.

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.

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X‐ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross‐β reflections characteristic of all amyloid fibrils. In addition to these cross‐β reflections, other reflections suggest the presence of well‐defined coiled‐coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full‐length apolipoprotein A1 with an apparent prevalence of the wild‐type species over the variant protein. We have found a ratio of full‐length wild‐type to mutant protein in plasma HDL of three to one. The polypeptide 1–93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C‐terminal end of the polypeptide.

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.

Folding and Fibrillogenesis: Clues from β2-Microglobulin

Renal failure impairs the clearance of beta(2)-microglobulin from the serum, with the result that this protein accumulates in joints under the form of amyloid fibrils. While the molecular mechanism leading to deposition of amyloid in vivo is not totally understood, some organic compounds, such as trifluoroethanol (TFE), are commonly used to promote the elongation of amyloid fibrils in vitro. This article gives some insights into the structural properties and the conformational states of beta(2)-microglobulin in the presence of TFE, using both the wild-type protein and the mutant Trp60Gly. The structure of the native state of the protein is rather insensitive to the presence of the alcohol, but the stability of this state is lowered in comparison to some other conformational states. In particular, a native-like folding intermediate is observed in the presence of moderate concentrations of TFE. Instead, at higher concentrations of the alcohol, the population of a disordered native-unlike state is dominant and correlates with the ability to elongate fibrils.

Molecular dynamics simulation of β2‐microglobulin in denaturing and stabilizing conditions

β2‐Microglobulin has been a model system for the study of fibril formation for 20 years. The experimental study of β2‐microglobulin structure, dynamics, and thermodynamics in solution, at atomic detail, along the pathway leading to fibril formation is difficult because the onset of disorder and aggregation prevents signal resolution in Nuclear Magnetic Resonance experiments. Moreover, it is difficult to characterize conformers in exchange equilibrium. To gain insight (at atomic level) on processes for which experimental information is available at molecular or supramolecular level, molecular dynamics simulations have been widely used in the last decade. Here, we use molecular dynamics to address three key aspects of β2‐microglobulin, which are known to be relevant to amyloid formation: (1) 60 ns molecular dynamics simulations of β2‐microglobulin in trifluoroethanol and in conditions mimicking low pH are used to study the behavior of the protein in environmental conditions that are able to trigger amyloid formation; (2) adaptive biasing force molecular dynamics simulation is used to force cis‐trans isomerization at Proline 32 and to calculate the relative free energy in the folded and unfolded state. The native‐like trans‐conformer (known as intermediate 2 and determining the slow phase of refolding), is simulated for 10 ns, detailing the possible link between cis‐trans isomerization and conformational disorder; (3) molecular dynamics simulation of highly concentrated doxycycline (a molecule able to suppress fibril formation) in the presence of β2‐microglobulin provides details of the binding modes of the drug and a rationale for its effect. Proteins 2011.