Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tailby Orly Avraham, Amit Meir, Alexander Fish, Edward A. Bayer, Oded Livnah

Journal of Structural Biology

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Year
2015
DOI
10.1016/j.jsb.2015.06.020
Subject
Structural Biology

Text

ith rd , The , The t, Isr

Received 12 May 2015

Received in revised form 22 June 2015

Accepted 24 June 2015

Available online xxxx

Keywords:

High affinity systems

Their dimeric configuration is a quaternary substructure of the classical tetrameric avidins which lacks fies their applications (Gonzalez et al., 1999; Pahler et al., 1987).

The structures of avidin and streptavidin consist of four identical subunits that form a homo-tetrameric assembly with four biotin-binding sites (Livnah et al., 1993; Weber et al., 1989). The topology of each monomer comprises an 8-stranded b-barrel with and strept er thereb stantially sealing the biotin-binding site (Chilkoti et al.,

Sano and Cantor, 1995). The lack of the critical Trp, via either genesis or dissociation of the tetramer, results in a subs decrease in affinity toward biotin (Chilkoti et al., 1995).

The exploitation of the (strept)avidin–biotin system became possible due to the conjugation of biotin via its carboxylate to various molecules and surfaces while maintaining the high affinity toward the avidins through its bicyclic ring system (Pazy et al., 2002). In addition to the high-affinity biotin derivatives, specific lower affinity peptides have been described that bind streptavidin ⇑ Corresponding author at: Department of Biological Chemistry, The Institute of

Life Sciences, The Wolfson Centre for Applied Structural Biology, The Hebrew

University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.

E-mail address: oded.livnah@huji.ac.il (O. Livnah).

Journal of Structural Biology xxx (2015) xxx–xxx

Contents lists availab

Journal of Struc .ealleled affinity toward biotin, avidin and streptavidin are both hyper-thermostable, a feature which further promotes and diversiwhere a Trp residue (110 and 120 in avidin respectively) crosses over to an adjacent monomhttp://dx.doi.org/10.1016/j.jsb.2015.06.020 1047-8477/ 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Avraham, O., et al. Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail. J. Struct. Biol. (2015), dx.doi.org/10.1016/j.jsb.2015.06.020avidin, y sub1995; mutatantial1. Introduction

Avidin–biotin based technology has been studied and utilized extensively over the past 4 decades. The high affinity between the avidins (mainly hen egg white avidin and bacterial streptavidin from Streptomyces avidinii) and their biotin ligand (Kd of 1014– 1016) (Green, 1975, 1990) has formed the basis for numerous biotechnological applications (Bayer and Wilchek, 1990; Laitinen et al., 2007; Wilchek and Bayer, 1990). In addition to their unparthe biotin-binding site at the wide end of the b-barrel. In the homo-tetrameric assembly there are 3 types of monomer–monomer interactions (Fig. 1a). The 1–3 interaction (numbered according to Livnah et al., 1993) has the smallest contact area between the two monomers and involves 3–4 amino acid residues from each monomer, depending on the avidin type. Interaction 1–4 has the largest contact surface area and is crucial for the stability of the quaternary structure (Kurzban et al., 1991; Livnah et al., 1993). Interaction 1–2 is critical for the high-affinity biotin bindingThermostability

Hoefavidin

Biotin

Peptide design

Structural biologythe requirement of the critical Trp that defines the tetramer and dictates the tenacious interaction with biotin. Hoefavidin, derived from the bacterium Hoeflea phototrophica DFL-43T, is the third characterized member of the dimeric avidin subfamily. Like the other members of this group, hoefavidin is a thermostable protein that contains a disulfide bridge between Cys57 and Cys88, thereby connecting and stabilizing the L3,4 and L5,6 loops. This represents a distinctive characteristic of dimeric avidins that compensates for the lack of Trp and enables their dimeric configuration. The X-ray structure of the intact hoefavidin revealed unique crystal packing generated by an octameric cylindrical structure wherein the

C-termini segments of each monomer is introduced into the entrance of the biotin-binding site of an adjacent non-canonical monomer. This anomaly in the protein structure served as a lead toward the design of specific binding peptides. We screened for specific hoefavidin binding peptides derived from the

C-terminal region and two peptides were obtained that bind a truncated form of hoefavidin (lacking the last 10 amino acids) with dissociation constants of 105 M. The crystal structure of short hoefavidin complexed with a C-terminal derived peptide revealed the mode of binding. These peptides may form the basis of novel and reversible binders for dimeric avidins.  2015 Elsevier Inc. All rights reserved.Article history: Dimeric avidins are a newly discovered subgroup of the avidin family that bind biotin with high affinity.Hoefavidin: A dimeric bacterial avidin w

Orly Avraham a, Amit Meir a, Alexander Fish b, Edwa aDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences

The Edmond J. Safra Campus, Jerusalem 91904, Israel bDivision of Biochemistry, The Netherlands Cancer Institute (NKI), 1066 CX Amsterdam cDepartment of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovo a r t i c l e i n f o a b s t r a c t journal homepage: wwwa C-terminal binding tail

A. Bayer c, Oded Livnah a,⇑

Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem,

Netherlands ael le at ScienceDirect tural Biology lsevier .com/locate /y jsbihttp:// uctu2 O. Avraham et al. / Journal of Strand are widely used for labeling and isolation purposes (Skerra and

Schmidt, 2000). These short peptides were initially discovered using phage display approaches and are specific toward streptavidin (Devlin et al., 1990). A streptavidin-binding peptide was first used in the detection and purification of antibodies, and was termed strep-tag (Schmidt and Skerra, 1993). Subsequently, strep-tag I was studied and manipulated further to generate strep-tag II, both of which have the consensus binding sequence of HPQ (His-Pro-Gln) (Devlin et al., 1990). The two peptides were shown to exhibit dissociation constants of 37 lM and 72 lM, respectively, and their crystal structures complexed with streptavidin were determined, thus revealing their common mode of binding (Schmidt et al., 1996). In order to further increase the binding affinity of the peptide, streptavidin was subjected to