Structure‐specific binding recognition of a methanogen chromosomal protein

Abstract

The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four-way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA double helix.Keywords : four-way junction; DNA bending ; architectural protein; archaea; MC1.Methanogen chromosomal protein (MC1) is the major chromosomal protein of various species of Methanosarcinaceae, a family of methanogenic archaea [1]. In the Methanosarcina sp. CHTI 55 strain, MC1 is a small protein of 93 amino acids (11 kDa) that contains a high amount of charged residues (24% basic residues and 14% acidic residues) and no large hydrophobic domain [2]. With respect to its primary structure, MC1 has no similarity with any other eukaryotic, eubacterial or archaeal structural proteins [3].MC1 binds double-stranded DNA as a monomer in a noncooperative way with a binding site size of about 11 bp [4]. It protects DNA against thermal denaturation [5] and against radiolysis by fast neutrons [6]. Previous studies have shown that MC1 binding induces bends in DNA. Indeed, MC1 is able to induce cyclisation of short DNA fragments with T4 DNA ligase [7], and sharp bends in DNA have been shown by electron and atomic force microscopy experiments [8,9]. MC1 has a quite strong affinity for bent and unwound DNA as supercoiled DNA minicircles [10]. It has been shown that even if MC1 has no absolute DNA-binding site, it can produce a discrete footprint on some sequences [11]. It is likely that these preferential bindings are due to readily deformable regions.Distorted DNA structures are present in some significant biological processes. Holliday structures are four-stranded DNA intermediates generated during recombination [12]. The global structure of these branched DNAs has been studied in detail using synthetic four-way DNA junctions [13Ϫ15]. In the absence of cations, the junction is extended in a square conformation, whereas in the presence of cations, the DNA folds into an X-shaped structure [16,17]. Two different isomers can exist depending on the base sequence at the centre of the junction [18,19].Various proteins exhibit structure-selective binding to DNA junctions. Some of these proteins are nucleases that cleave these junctions (for a review see [20]). Others proteins that only bind to these junctions have also been identified, e.g. structural proteins similar to different kinds of HMG proteins, the Escherichia coli protein HU and the linker histones bind with high selectivity to four-way DNA junctions ...

Refined solution structure and backbone dynamics of the archaeal MC1 protein

“…An antiparallel b-bulge (B1), composed of Leu8, His16, and Gly17, is present in seven structures, and another antiparallel b-bulge (B2), composed of Val57, Glu87, and Arg88, is observed for all the structures. The secondary structure elements are connected by loops LP1 (10)(11)(12)(13)(14), LP2 (22)(23)(24), LP3 (35)(36)(37)(38)(39)(40)(41)(42), LP4 (50-53), and LP5 (67-77), referred to as 'arm' in the text. The latter now appears to be remote from the protein core, whereas it was previously described as pulled down on the a-helix.…”

“…One water molecule bridging NH(Glu87) and CO(Val57) through hydrogen bonding contributed to these dynamics. Nanosecond slow motions observed in loops LP3 (35)(36)(37)(38)(39)(40)(41)(42) and LP5 (67-77) reflected the lack of stable hydrogen bonds, whereas the other loops, LP1 (10)(11)(12)(13)(14), LP2 (22)(23)(24), and LP4 (50-53), were stabilized by several hydrogen bonds. Dynamics are often directly related to function.…”

Archaeal chromatin proteins

“…Sequence comparison reveals no relationship between MC1 and other known chromatin proteins. MC1, estimated to be present at ~1 monomer per ~170 bp in the Methanosarcina cell [28], binds preferentially to negatively supercoiled DNA and to four-way Holliday structures with a binding size of ~11 bp per monomer [30]. Binding by MC1 results in DNA bending and negative DNA supercoiling, and protects the DNA against radiolysis and heat denaturation [28].…”

Archaeal chromatin and transcription

“…MC1 family members are also predicted to be present in Halococcus , Halobacterium and Haloferax species. MC1 binds preferentially to negatively supercoiled DNA and to four‐way Holliday structures with one monomer calculated to interact with ∼11 bp of double‐stranded DNA (Paradinas et al ., 1998). Binding is non‐co‐operative, involves residues near the C‐terminus of the protein, results in DNA bending and negative supercoiling, and protects DNA against radiolysis and heat denaturation.…”