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To gain better understanding of the impact of the mutations identified in section 3.3, their position was determined in relation to the known LPL functional domains. We used the published tridimensional model of PL (Winkler et al., 1990) on which key LPL structures had been superimposed (Santamarina-Fojo, 1992). The true position of the residues in native LPL may differ slightly from this representation. The position of disulfide bonds, the heparin-binding domain (aa292-302) and the active-site triad (Ser 132, Aspl56, His241) were used as guides, along with schematic diagrams of conserved and variable elements (Derewenda and Cambillau, 1991). To determine further whether the residues were located on the outside of the protein or buried within, the ribbon PL model drawn by Lalouel et al. (1992) based on the Winkler structure proved to be very useful.

The nine point mutations causing amino acid substitutions were positioned on the PL model structure represented on Fig.3.8. All the mutations were located in the large, catalytic N-terminal domain (aa 1-334). Recent data reviewed by Lalouel et al. (1992) and presented by Derewenda and Cambillau (1991) suggests that the tightly packed, central jS-sheet and a-helices of this domain are very sensitive to alterations. Therefore, even apparently conservative mutations in this region are likely to affect LPL function.

Trp86 has its bulky side chain apparently packed against a short stretch of hydrophobic residues, Ala98-Gly99-TyrlOO. The residue is conserved in human HL, PL and LPL and a Trp to Arg substitution at this position has been shown to inactivate the enzyme (Ishimura-Oka et al., 1992a). Trp86 is also very close to one of three active site loops (Gln91 - Pro95) thought to be involved in substrate binding (van Tilbeurgh et al., 1994) and may itself interact with the acyl chain of the triglyceride. Although its effect

may be expected to be less severe than W86R, it is possible that the replacement of tryptophan by glycine may force a rearrangement of hydrophobic residues in the vicinity or disrupt van der Waals interactions. This may in turn change the orientation of the substrate-binding loop and leading to less efficient presentation of the substrate.

Residue 158 appears to be located in a fold, in close proximity to both Aspl56 and Ser 132, two members of the catalytic triad. A lai58 is part of a proposed substrate- binding loop (residues 157-160) and may display hydrophobic interactions with the triglyceride substrate (van Tilbeurgh et al., 1994). Alal58 is also part of a strictly conserved segment in all lipases (TGLDPA) (Derewenda and Cambillau, 1991; Bensadoun, 1992) and other substitutions in this region at positions 154, 156 zmd 157 have been shown to completely abolish activity (Ma et al., 1992a, Bruin et al., 1992, 1993). Therefore, the presence of the polar threonine residue at this position is expected to markedly affect LPL function, probably by hindering the access of the substrate to the catalytic site or by weakening the strength of the binding. Alternatively, the hydroxyl group of Thr 158 may interfere with the charge relay system of the triad by disrupting the alignment of Serl32, Aspl56 and His 241. However, this possiblity is difficult to evaluate in the absence o f precise information regarding the orientation of the Thr side chain.

I n t e r f a c i a l b i n d i n g s i t e ' l A s n Lid 1 3 2 - H e p a r i n b i n d i n g s i t e ^ F i g . 3 . 8 P o s i t i o n i n g o f t h e L P L g e n e m u t a t i o n s i d e n t i f i e d in t h i s s t u d y o n t h e 3 D m o d e l o f h u m a n P L ( W i n k l e r et a ! . , 1 9 9 0 ). T h e m u t a t i o n s are in d ic ate d by s h a d e d d o ts on the s tr u c t u re , n e x t to t h e o n e - l e t t e r c o d e f o r ea ch m u t a t i o n , in r e la tio n to th e c a ta ly tic triad a m in o acids (dots w ith c r o s s e s ) an d th e g l y c o s y l a t i o n s ite ( A s n 4 3 ). In tern al h y d r o p h o b i c s e g m e n t s w h e r e m u ta tio n s h a v e b e e n id e n tifie d a r e s h o w n b y a d i s c o n t i n u o u s line (s h o rt d a s h e s ) w h e r e a s the d is u l fid e b r i d g e s a r e in d ic ate d by lo n g d a s h e s .

The H183Q substitution was recently identified in a proband of Swiss/Russian descent (Tenkanen et al., 1994) and shown to lead to an inactive enzyme. This residue appears to be in close contact with the helix containing His241 in a densely packed region. Haubenwallner et al. (1993) have recently reported that a conservative substitution with respect to charge but not to size at position 180 (Asp to Glu) abolishes activity, probably due to steric hindrance. By examining the helix sequence in the vicinity of His 183 and assuming compatible orientation of side chains, it is possible to envisage an ionic interaction with Glu242. This bond would be disrupted by the substitution with Gin and may alter the topology of the catalytic triad.

Residues 188 and 193 are located on the third proposed substrate-binding loop element (Arg 187 - Ilel96), under the lid structure which is predicted to rotate away upon interfacial activation. Although He 194 is the only residue thought to interact directly with the substrate (van Tilbeurgh et al., 1994), it has been proposed that the loop segment forms a hydrophobic groove. If this model is correct, the replacement of serine by the bulkier, charged asparagine residue would be expected to effect a major change in the local structure and restrict substrate access to the catalytic site. By analogy with the analysis of Derewenda and Cambillau (1991) for the G188E mutation, electrostatic interactions with Arg 187 and Glu227 (part of the lid structure) can be postulated. These interactions could modify the shape of the protein such as to make the LPL dimer unstable, as the G188E and G195E substitutions have been shown to do (Hata et al.,

1992). Alternatively, the attraction to Glu227 may increase the rigidity of the structure and thus interfere with the displacement of the lid covering the catalytic site.

Amino acid positions 207, 301 and 303 are not in close proximity to catalytic triad residues but rather are part of the last two parallel /8-strands of the N-terminal

domain. The closely packed jS-sheet structure of the N-terminal provides the stable frame needed to maintain the alignment o f the catalytic triad residues. The substitutions at positions 301 and 303 both involve the replacement of bulky hydrophobic residues by smaller and less hydrophobic (in the case of Thr) ones, which may lead to weaker interactions between strands. This loss of rigidity of the j3-sheet structure may perturb the linear arrangement of the catalytic triad. For its part, the substitution at position 207 involves the replacement of a proline residue, an imino acid which can adopt a limited number o f conformations (MacArthur and Thornton, 1991). The presence o f a leucine residue may introduce some unwanted flexibility in the region, again possibly disturbing the delicate alignment of the triad.

Finally, residue 291 is located on a protruding loop extending away from the catalytic site in the PL model. However, this segment (aa290-300 in LPL) is poorly conserved between LPL and PL so that its structure in LPL may not be accurately predicted by the PL model (Persson et al., 1989, Derewenda and Cambillau, 1991). The residue is still likely to be on the surface of the normal protein because asparagine has a polar side chain. Moreover, it is surrounded by positively charged residues from two segments which are involved in heparin-binding (aa279-283 and 292-300) (Hata et al., 1993). Ma et al. (1993c) recently reported that the Asn to Ser substitution had half­ normal activity in vitro. Residue 291 does not appear to lie in close proximity to the active site so that its effect on enzyme activity might be indirect. It is also possible that altered hydrogen bonding decreases the stability of the LPL dimer.

The 2-nt deletion in exon 6 is expected to result in a truncated protein lacking part of the catalytic domain (aa 254-334) including a putative heparin-binding site and

the whole C-terminal lipid binding domain. This protein could not be anchored and stabilised on the cell surface via interactions with HSPG and would probably be rapidly degraded.