Crystal structure of L68V mutant of human cystatin C, an amyloidogenic protein

Abstract

Human cystatin C (wt cystatin C) is a one-chain protein (13,343 Da, 120 amino acids) that reversibly inhibits cysteine proteases of the papain and legumain families [1]. Besides its inhibitory function, hCC plays a causal role in development of one of neuropathological diseases - an amyloid angiopathy. In brain arteries of elderly individuals suffering from this disease cystatin C forms massive amyloid deposits leading to cerebral hemorrhages and finally death of patients [2]. The naturally occurring single point mutant of human cystatin C - Leu68Gln hCC - is implicated in hereditary cystatin C amyloid angiopathy, also known as hereditary cerebral hemorrhage with amyloidosis, Icelandic type. L68Q variant oligomerizes much more easily than its wild-type analog, even at physiological temperature. Deposition of L68Q aggregates in cerebral and spinal arteries and arterioles leads to recurrent hemorrhagic strokes causing serious brain damage and death of young, less than 40 years old, adults [3]. The increased propensity of the L68Q hCC variant for oligomerization can be connected with its decreased conformational stability caused by the introduction of a bulky and polar residue into the hydrophobic interior of the protein [4]. To get deeper insight into the possible mechanism of hCC oligomerization and asses the impact of modifications introduced into position 68 on this process we designed and constructed hCC variants with Leu68 residue replaced isosteric but polar Asn residue and hydrophobic but smaller in the van der Waals radius valine. The first mutation resulted in strong destabilization of the protein comparable with the one caused by glutamine residue. Valine mutant turned out to be more stable and could be expressed in good yield as a monomeric protein, but it shows increased propensity for dimerization in in vitro tests. Since we were able to obtain well diffracting crystals of the hCC L68V variant at two crystallization conditions (pH=4.6 and pH=8.0), the properties of this mutant will be discussed in connection to the obtained structural data.