Cold-Shock Domains-Abundance, Structure, Properties, and Nucleic-Acid Binding

Abstract

The cold-shock domain has a deceptively simple architecture but supports a complex biology. It is conserved from bacteria to man and has representatives in all kingdoms of life. Bacterial cold-shock proteins consist of a single cold-shock domain and some, but not all are induced by cold shock. Cold-shock domains in human proteins are often associated with natively unfolded protein segments and more rarely with other folded domains. Cold-shock proteins and domains share a five-stranded all-antiparallel β-barrel structure and a conserved surface that binds single-stranded nucleic acids, predominantly by stacking interactions between nucleobases and aromatic protein sidechains. This conserved binding mode explains the cold-shock domains' ability to associate with both DNA and RNA strands and their limited sequence selectivity. The promiscuous DNA and RNA binding provides a rationale for the ability of cold-shock domain-containing proteins to function in transcription regulation and DNA-damage repair as well as in regulating splicing, translation, mRNA stability and RNA sequestration.

Keywords: OB fold; RNA-binding domain; Y-box binding protein; cold-shock domain; cold-shock protein; domain fold; gene regulation; nucleic-acid binding; protein stability and folding; protein structure.

Figure 1

Sequence alignment of representative bacterial CSPs and CSDs from human proteins. The Xenopus laevis FRGY1 and FRGY2 (YBOX1, YBX2A, YBX2B) proteins are also included. Human CSDE1 contains five CSDs, all other proteins contain or consist of a single CSD. Proteins are identified by their Uniprot [22] entry number and name. The secondary-structure annotation atop the sequence follows BsCspB, the first CSP for which a crystal structure was determined [23]. Residues conserved across all aligned CSDs are highlighted on dark blue background and shown with capital letters in the consensus sequence. Residues conserved in ≥50% of the sequences are shown on a light blue background and with lower-case letters in the consensus. Sequences were aligned using the Clustal Omega server [24]. The sequence motifs RNP1 ([YF]-G-F-I) and RNP2 ([YF]-[YF]-H) are associated with RNA binding and indicated according to Prosite [25].

Figure 2

Proteins with cold-shock domains. Domain annotations for one representative bacterial CSP and human CSD-containing proteins according to SMART [27]. For CSDE1, Pfam [31] agrees with the domain annotation shown here. Uniprot [22] annotates two additional CSDs in CSDE1, one between CSD3 and CSD4 and one between CSD4 and CSD5, as well as two additional truncated CSDs, one between CSD1 and CSD2 and one between CSD2 and CSD3. InterPro [32] annotates a total of nine CSDs in CSDE1, those shown here and four additional CSDs filling the gaps. CSDs are displayed as green diamonds labeled “CSP”, the stunted CCHC-type zinc fingers (zinc knuckles) present in the LIN28 proteins as blue vertical bars, and low-complexity sequences as pink bars. Proteins are drawn to scale.

Figure 3

Three-dimensional structure of cold-shock proteins and domains determined at near-atomic resolution. (a) Cartoon drawing, (b) topology diagram with β-sheet stabilizing hydrogen bonds, and (c) electrostatic surface potential colored from red (−10 kT/e) to blue (+10 kT/e) of BcCspB (PDB entry 1c9o). (d) Schematic drawing of the X. tropicalis LIN28 CSD (PDB entry 3ulj). Orthogonal views are presented in (a,c). (e) Conserved residues on the surface of StCspE (PDB entry 3i2z). RNP1/RNP2, RNA-binding motifs [20]. Note the close structural similarity between the bacterial BcCspB [97] and the eukaryotic LIN28B CSD [100], the separation of negative (red) and positive (blue) surface charge in (c) and the asymmetric distribution of conserved residues over the CSP surface. Cartoon drawings were prepared with PyMOL [101], the topology diagram is based on PDBsum [102], and the electrostatic surface was calculated with the Adaptive Poisson-Boltzmann Solver (APBS) plugin [103] of PyMOL.

Figure 4

Domain- and segment-swapped forms of the cold-shock protein. (a) BcCspB domain-swapped dimer ([113], PDB entry 2hax). Protein-bound DNA strands were omitted for the sake of clarity. (b) Crystal structure of EcCspA ([95], PDB entry 1mjc) and (c) NMR structure of the S1 domain of E. coli polynucleotide phosphorylase ([117], PDB entry 1sro). Strands β1-β3 of both proteins that recombine to form 1b11 are highlighted by darker colors. (d) Structure of combinatorial protein 1b11 ([118], PDB entry 2bh8). Drawings prepared with PyMOL [101].

Figure 5

DNA single strands bound to CSDs. (a) Solvent-accessible surface and (b) cartoon drawing of BcCspB bound to (dT)₆ ([113], PDB entry 2hax). Only one globular unit formed by two strands of a domain-swapped BcCspB dimer (see Figure 4) is displayed. (c) (dT)₇ bound to LIN28B CSD ([100], PDB entry 4a76). Note how stacking interactions between nucleobases and aromatic amino-acid sidechains contribute prominently to the closely similar binding interfaces of the bacterial CSP and the eukaryotic CSD. Drawings prepared with PyMOL [101].

Figure 6

RNA single strands bound to CSDs. (a) GUCUUUA bound to BsCspB ([139], PDB entry 3pf4). The 5′ and 3′-terminal nucleotides of the co-crystallized RNA strand are not revealed in the structure. (b) UCAUCU bound to the YBX1 CSD ([11], PDB entry 5ytv). The 5′ and 3′-terminal nucleotides of the co-crystallized RNA strand are not revealed in the structure. (c) The microRNA precursor preE_M-let-7d bound to the CSD and ZKD of mouse LIN28A ([140], PDB entry 3trz). Drawings prepared with PyMOL [101].