Tài liệu Báo cáo Y học: Structural and biochemical characterization of neuronal calretinin domain I–

Structural and biochemical characterization of neuronal calretinin

domain I–II (residues 1–100)

Comparison to homologous calbindin D28k domain I–II (residues 1–93)

Małgorzata Palczewska1

, Patrick Groves1

, Attila Ambrus2,*, Agata Kaleta1

, Katalin E. Ko¨ ve´r

3

, Gyula Batta4

and Jacek Kuz´nicki1,5

1

Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland;

2

Department of Biochemistry and Molecular Biology, University of Debrecen, Hungary; 3

Department of Inorganic Chemistry,

and 4

Research Group for Antibiotics, Department of Chemistry, University of Debrecen, Hungary; 5

International Institute of Molecular and Cell Biology, Warsaw, Poland

This study characterizes the calcium-bound CR I–II

domain (residues 1–100) of rat calretinin (CR). CR, with

six EF-hand motifs, is believed to function as a neuronal

intracellular calcium-buffer and/or calcium-sensor. The

secondary structure of CR I–II, defined by standard NMR

methods on 13C,15N-labeled protein, contains four helices

and two short interacting segments of extended structure

between the calcium-binding loops. The linker between the

two helix–loop–helix, EF-hand motifs is 12 residues long.

Limited trypsinolysis at K60 (there are 10 other K/R

residues in CR I–II) confirms that the linker of CR I–II is

solvent-exposed and that other potential sites are protected

by regular secondary structure. 45Ca-overlay of glutathione

S-transferase (GST)–CR(1–60) and GST–CR(61–100)

fusion proteins confirm that both EF-hands of CR I–II have

intrinsic calcium-binding properties. The primary sequence

and NMR chemical shifts, including calcium-sensitive glycine

residues, also suggest that both EF-hand loops of CR I–II bind

calcium. NMR relaxation, analytical ultracentrifugation,

chemical cross-linking and NMR translation diffusion

measurements indicate that CR I–II exists as a monomer.

Calb I–II (the homologous domain of calbindin D28k) has the

same EF-hand secondary structures as CR I–II, except that

helix B is three residues longer and the linker has only four

residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald,

P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur.

J. Biochem. 262, 933–938]. In contrast, Calb I–II binds one

calcium cation per mono-meric unit and exists as a dimer.

Despite close homology and similar secondary structures,

CR I–II and Calb I–II probably have distinct tertiary

structure features that suggest different cellular functions for

the full-length proteins.

Keywords: calretinin; calcium; calbindin D28k; EF-hand;

NMR secondary structure.

Calretinin (CR) and calbindin D28k (Calb) are homologous

calcium-binding EF-hand proteins with 59% sequence

identity (rat forms) [1–3]. The proteins contain six

helix–loop–helix motifs (EF-hands) in which the loops

carry the calcium-binding ligands. The rat sequences are

271 (CR) and 261 (Calb) amino acids long [4,5]. Both

proteins have distinct, predominantly neuronal cellular

distributions [3,6–9] and Calb is additionally found in the

digestive system [10]. Both proteins are used as markers for

a subset of neurons and several neurodegenerative diseases

[11–13]. Immunohistochemistry of CR is used to distin￾guish adenocarcinomas from mesothelioma [14,15], with

CR playing a role in the early stages of mesothelioma

[16]. In contrast, Calb is found in subpopulations of neuro￾endocrine phenotypes of some carcinoids and small-cell

carcinomas [17].

EF-hand proteins act as intracellular calcium-sensors,

linked to protein signaling cascades, and/or calcium-buffers

[3]. For example, calmodulin binds to more than 100 different

proteins in a calcium-specific manner, while parvalbumin is

considered important in maintaining nontoxic levels of free

intracellular calcium through its buffering ability [3]. The

function of CR is unclear 2 there is evidence for both

buffer and sensor roles (reviewed in [18]) but no definite

target protein has yet been identified for CR to support a

sensor role. Calb appears to play a buffer role in neurons

[19] and possibly facilitates calcium uptake through the

digestive system [10]. However, there is evidence that

intestinal brush border membrane alkaline phosphatase

Correspondence to J. Kuznicki, Department of Molecular and Cellular

Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur

Street, 02-093 Warsaw, Poland. Fax: 1 48 22 822 53 42,

Tel.: 1 48 22 659 31 43, E-mail: [email protected] or to G. Batta,

Research Group for Antibiotics, Department of Chemistry, Egyetem te´r.

1, University of Debrecen, PO Box 70, H-4010 Debrecen, Hungary.

Fax: 1 36 52 512 914, Tel.: 1 36 52 512 900 extn 2370,

E-mail: [email protected]

*Present address: The University of Arizona, Department of Chemistry,

1306 E University, Tucson, AZ 85721–0041, USA.

Note: a web page is available at

http://www.nencki.gov.pl/labs/cbplab/kuzhome.htm

(Received 28 June 2001, revised 24 September 2001, accepted

4 October 2001)

Abbreviations: CR, calretinin (rat isoform, unless otherwise stated); CR

I–II, calretinin residues 1–100 consisting of the first pair of EF-hand

motifs; Calb, calbindin D28k (rat isoform, unless otherwise stated); Calb

I–II, calbindin D28k 1–93 consisting of the first pair of EF-hand motifs;

Calb III–IV, calbindin D28k 79–193, consisting of the second pair of

EF-hand motifs; CSI, chemical shift index; GST, glutathione

S-transferase; DOSY, diffusion ordered spectroscopy.

Eur. J. Biochem. 268, 6229–6237 (2001) q FEBS 2001