Effects of His mutations on the fibrillation of amyloidogenic Vk6 protein Wil under acidic and physiological conditions Tomonori Mishima a , Takatoshi Ohkuri a , Akira Monji b , Takaaki Kanemaru c , Yoshito Abe a , Tadashi Ueda a, * a Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan b Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan c Morphology Core Unit, Kyushu University Hospital, Fukuoka 812-8582, Japan article info Article history: Received 31 October 2009 Available online 22 November 2009 Keywords: Amyloid Histidine NMR relaxation Residual structure summary Recently, we showed that the recombinant (r) Vk6 protein Wil exhibits a more disrupted residual struc- ture and a longer lag time for fibril formation than the rVk6 protein Jto under highly unfolding conditions at pH 2. Here, we focused on the roles of three histidine residues specific for Wil, which are positively charged at pH 2 and could repel one another. Heteronuclear relaxation experiments revealed that a mutant Wil with H34Q, H53Q and H93S mutations (3HmutWil) had larger R 2 values only in the region of residues 22–55 and formed fibrils much earlier than Wil at pH 2. 3HmutWil also showed a decrease in ThT fluorescence intensity compared with Wil in fibrillation experiments at pH 7.5. The present results suggest that these three histidine residues play important roles in the fibrillation of Wil at both pH 2 and pH 7.5. Ó 2009 Elsevier Inc. All rights reserved. Introduction A number of human diseases such as Alzheimer’s, Parkinson’s and Creutzfeldt–Jacob disease originate from the accumulation of misfolded and aggregated proteins [1–3]. Although toxic proteins related to these diseases do not exhibit special characteristics in terms of their three-dimensional structures or amino acid se- quences [4], all are known to form fibrillar aggregates, termed amyloid fibrils. However, many non-disease-related proteins are also able to form amyloid-like fibrils under appropriate conditions, suggesting that amyloid fibrillation is a generic property of poly- peptide chains [5]. AL amyloidosis is a monoclonal plasma cell dis- order associated with the overproduction and extracellular deposition of light chains in the form of insoluble fibrils [6,7]. Although other amyloidogenic proteins may be wild-type or linked to a single hereditary mutation, an abundance of mutations is char- acteristic of AL amyloidosis. It has been found that the k6 subtype of light chains is preferentially associated with AL amyloidosis [8], resulting in interest in the characterization of k6 light chains. The crystal structures of two recombinant light chain variable domains belonging to the k6 subtype (rVk6), the amyloidogenic Wil and nonamyloidogenic Jto, have been reported [9]. Wil and Jto are acidic proteins composed of 111 amino acid residues and contain a single disulfide bond between Cys23 and Cys88 (Fig. 1A). It has been reported that Wil is thermodynamically less stable and con- sequently forms fibrils with a shorter lag time than Jto at pH 7.5 [10]. In addition, Wall et al. showed that a long-range electrostatic interaction between Asp29 and Arg68, which is present in Jto but not in Wil, contributes to the increased stability and decreased amyloidogenicity of Jto [11]. However, the mechanisms behind these differences between Jto and Wil are not fully understood. Recently, we showed that Jto and Wil have non-random resid- ual structures in regions that include some hydrophobic residues and a single disulfide bond at pH 2 and 37 °C [12]. The residual structure of Wil is more disrupted than that of Jto. Interestingly, Wil required a longer time to form fibrils than Jto under the same conditions with constant stirring. These results suggest that the maintenance of a residual structure is necessary for fibrillation of rVk6 proteins at pH 2. Since a lag time in the fibrillation process was reported to correspond to the formation of fibril nuclei [13,14], the residual structure is likely to be particularly important for nucleation in the fibrillation process. The differences in the residual structure and fibrillation can be attributed to the sequence of Wil, which differs from the sequences of k6a and Jto at 10 and 18 positions, respectively (Fig. 1A). In particular, Wil has three addi- tional histidine residues, His34, His53 and His93, which would be positively charged at pH 2. Therefore, it is possible that these positively charged residues repel one another, thereby resulting 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.11.108 Abbreviations: CD, circular dichroism; EM, electron microscopy; HSQC, heter- onuclear single quantum coherence; r, recombinant; R 2 =(T 2 ) À1 , transverse relax- ation rate; ThT, thioflavine T; V L , light chain variable domain; Vk6, lambda 6 variable domain * Corresponding author. Fax: +81 92 642 6667. E-mail address: ueda@phar.kyushu-u.ac.jp (T. Ueda). Biochemical and Biophysical Research Communications 391 (2010) 615–620 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc