RICERCA ITALIANA     

Research program

Identification of folding and misfolding determinants by site-directed mutagenesis.

University Co-ordinator

Università degli Studi di UDINE - SCIENZE E TECNOLOGIE BIOMEDICHE - ()

Research Unit Leader

Gennaro Esposito

Description

The applicant groups have been involved, over the last few years, in extensive studies on b2m, using biochemistry and molecular biology as well as a series of biophysical approaches such as mass spectrometry, limited proteolysis, NMR and AFM which appear particularly well suited for this specific biological problem. Our group, in particular, was involved in the NMR spectroscopy and modelling part. The results of the structural analysis performed on b2m, its mutants and fragment devoid of the N-terminal hexapeptide (DN6b2m) have been summarized above. The outlined picture, based on the NMR structural conclusions, suggests that DN6b2m could be regarded as an amyloidogenic conformation (Esposito et al., 2000; Verdone et al., 2002; Esposito et al., 2005). The results on fibril proteolysis show that the cleavage pattern is identical for either the intact protein fibrils and the fibrils formed by DN6b2m, and also that the latter fragment is produced from the proteolysis of intact b2m (Monti et al., 2002), which may account for the massive in vivo occurrence of the fragment in fibrils. Fibrils originate from the entire protein, once its native conformation is destabilized by, or sequestered by competition with, an amyloidogenic conformation that should be similar to the one described for DN6b2m.

Previous attempts of rational mutant design
The rationale that led us to recognise the prodromes of b2m amyloid transition was put at work to design mutants and variants. In particular, the crucial role for stability of H31 prompted us to project a mutant that, while keeping the aromatic character of the side chain, could not be charged around neutral pH. According to expectations, the H31Y mutant, where a tyrosine replaces histidine, is more stable than wild-type species by 1.5 kcal/mol, whereas dropping the aromatic side chain by substituting a serine for histidine stabilizes only by 0.6 kcal/mol (Corazza et al., 2004). The structure of H31Y mutant, determined by X-ray and NMR (Rosano et al., 2004), shows that i) the conformational closeness of H31Yb2m and wild-type protein, when the latter is either complexed in MHC-I crystal (Bjorkman et al., 1987) or isolated in solution (Verdone et al., 2002), but not when isolated in the crystal (Trinh et al, 2002), and ii) the heterogeneity in H31Y b2m crystal that also includes an alternative minor conformation where the N-terminal strand (strand A) is displaced from its native location, in agreement with the proposed evolution towards the amyloidogenic conformation. Besides predicting more stable mutants based on the critical residue H31, the structural information on the solution structure of b2m could be used to make a guess for less stable mutants than natural sequence. For instance, by suppressing the charge and the long side-chain of R3 one should obtain a less stable mutant, which is indeed found on replacing R3 with an alanine. The R3A mutant is destabilised by 0.4 kcal/mol with respect to wild-type b2m, but its NMR solution structure, however, looks still very similar to that of the parent sequence, with some dispersion increase within the conformational ensemble at DE and FG loops (Corazza et al., 2004). While suppression of the positive charge in position 31 for H31Y mutant mainly affects the electrostatic contribution to folding free energy, suppression of the positive charge in position 3 for R3A mutant affects mostly the corresponding solvation contribution (Corazza et al., 2004). Solvation free energy should likewise contribute most of the 1.9 kcal/mol stability decrease of DN3b2m, the truncated b2m variant devoid the N-terminal tripeptide, although in this case the actual lack of a sequence fragment impairs direct comparison between full-length sequence and truncated species. In addition, the presence in DN3b2m of a N-terminal methionyl residue, coming from expression in E. coli, places the backbone N-terminus ammonium in position 3 and hence does not totally suppress the positive charge at that location. This may account for the substantial conservation of the tertiary folding in DN3b2m inferred from the relative invariance of the NMR chemical shifts with respect to full-length b2m, at variance with the extensively destructured DN6b2m (Esposito et al., 2000) which proves also the most destabilised variant of b2m (2.5 kcal/mol).
For b2m and variants, the intermediate research led to identify a slow converting form on the pathway of refolding named I2 (Chiti et al 2001; Corazza et al., 2004) The kinetic lability of I2 was shown to be inversely related to the thermodynamic stability of the fully folded final product. Based on NMR evidence, in general the equilibrium population of I2 was rather low except for the truncated sequence DN3b2m. In this case, I2 was seen to account for some 25% of the overall protein concentration at 298 K.

Overall strategy outline
Based on previous results it is clear that fundamental multidisciplinary research is necessary to assess the determinants of structural misfolding leading to b2m amyloid. In addition to basic structural and functional investigation tools, amyloidogenic proteins can be studied by rapid fibrillogenesis methods at neutral pH that do not entail extreme conditions, such as low pH, concentrated metal ions, etc. By exploiting hydrophobic tuning of stability or electrostatic short range ordering, it is possible to obtain b2m fibrils (Yamamoto et al., 2004a; 2004 b; Myers et al., 2006; Relini et al., 2006). A screening of the fibrillogenic propensity of a wide range of b2m point mutants, systematically designed to map the whole sequence, is proposed to dissect the individual residue contributions to amyloidogenic propensity. The applicant groups use established experimental protocols and methodology to study different aspects of the biochemistry and biophysics of b2m. Expression and purification of the products will be performed by Pavia unit. The same unit will also determine the thermodynamic stability of the products, through chemical denaturation spectroscopic measurements, and the fibrillogenesis rate, through two different protocols, namely i) seeding at acidic pH , ii) seeding in the presence of trifluoroethanol. The solvent accessibility and the structural flexibility will be investigated by limited proteolysis studies by the Naples unit. The heterogeneous fibrillogenesis onto collagen fibres will be studied by AFM, along with the morphological characterization of the deposits by the Genoa unit. The kinetics of folding, unfolding and refolding will be studied by stopped-flow by the Florence unit. The NMR structural and dynamic characterisation of the products and the modeling calculations thereof will be performed by the Udine unit (the relative specific tasks will be detailed in the following). Finally the Florence unit will collect all the data and experimental parameters from the whole project team in order to establish a general parametric algorithm that may provide predictions and insights into b2m misfolding and fibrillogenesis.

Mutant design criteria
In order to devise an operative list for a b2m mutation plan other than a systematic generalised substitution of Ala residues for any position of the molecule, a few criteria were adopted to restrict the mutation sites. These criteria are:
i) mutations should involve those residues with the largest exposure area in the natively folded conformer;
ii) mutations should concern those residues with the largest exposure surface ouside strands A(I) and G (VIII), i.e. the two strands most likely involved in b2m partial unfolding;
iii) mutations should occur at those residues that, based on real-time 1D NMR evidence, are observed to change throughout the slow refolding phase (L23, I35, V37, L40).
According to the above criteria, one obtains a rather homogeneous distribution of mutation sites over the whole b2m sequence, although not really as regular as that obtained by applying a mutation every other three residues. The majority of mutations (18 out of 30) involves charged residues. A few consecutive mutation sites have been retained, e.g. K58 and D59 because these residues belong to the critical D-E loop and should establish an important interaction with the facing B-C loop. About ten among the selected residues are involved in the interaction with MHC-I heavy chain. Overall 30 mutations were selected (in addition to the mutants at position 3 and 31 already addressed) according to the following scheme:





Specific experimental tasks
The Udine unit will be primarily involved in the NMR characterisation of the different expressed mutants of b2m. A detailed conformational determination will not be possible for all the products. Selection of the mutants that will be worth submitting to detailed structural study will be performed based on the information provided by the other units.
The NMR techniques that will be employed for these investigations are the standard ones for proton and heteronuclear 2D and 3D spectroscopy supported by restrained molecular modeling. In particular we plan the acquisition of 2D 1H TOCSY, NOESY and COSY with double and triple quantum filters, 1H-15N correlation via SQC and MQC with and without 1H-relayed TOCSY and NOESY steps in 2 or 3 dimensions. Some of these experiments require 15N-labelled samples that will be purchased from ASLA (Riga, Latvia), once a satisfactory expression protocol will be available.
Gross structural comparisons between different mutants may rapidly be obtained by analyisis of the paramagnetic attenuation pattern measured in the presence and absence of a spin label (Esposito et al., 1992) or even from the differential water accessibilty pattern (Dalvit, 1996, 1998).
In addition to basic structural characterization, NMR spectroscopy can be used to measure the molecular mobility parameters via 15N relaxation studies. Comparative relaxation measurement may provide quick insights of the profiles of segmental mobilities between different mutants.
NMR can also provide very detailed information on the stability of secondary structure by measurements of the isotope exchange rates. These rates can be determined either far from denaturing conditions, where uncorrelated exchange predominates (EX2)and thermodynamic parameters on local stability are obtained, and close to denaturing conditions when correlated echange is observed to assess the kinetics of the denaturation process (EX1). Electrospray ionization mass spectrometry determination will complement the NMR determination and will help especially to investigate fast exchange processes.
An important part of the experimental NMR analysis will concern the so-called real time NMR determinations whereby we were able to measure the kinetics of the slow refolding intermediates of several b2m variants and infer structural conclusions about the nature of the slow-evolving intermediates. This type of determination is performed by NMR through continuous monitoring of a GdnCl denatured protein solution that is suddenly diluted with a renaturing buffer. We plan to perform this type of determinations with all the mutants that will be submitted to NMR analysis. Under favourable circumstances, a mutant may exhibit also slow unfolding intermediates that may be investigated by 1D NMR and compared with the pattern obtained from the corresponding refolding experiment. The same type of determinations may be performed in the presence of the heavy chain component of MHC-I, in particular when the mutation being examined involves a residue known to contact the heavy chain. The very presence of the heavy chain in the refolding buffer may alter the refolding rate if the unfolded conformation exhibit some affinity for the heavy chain. The extent of interaction between light and heavy chain may be analysed by classical tranfer NOE experiments (Clore and Gronenborn, 1982) performed under steady state conditions.
Any result obtained from structural and dynamic NMR studies will be made available to all other units, and in particular to the Florence unit that will assemble all the evidence from the systematic scrutiny proposed by the present reasearch project to identify general correlation for misfolding and amyloidogenesis determinants of b2m .