Expanding the proteome: disordered and alternatively folded proteins

Abstract

Proteins provide much of the scaffolding for life, as well as undertaking a variety of essential catalytic reactions. These characteristic functions have led us to presuppose that proteins are in general functional only when well structured and correctly folded. As we begin to explore the repertoire of possible protein sequences inherent in the human and other genomes, two stark facts that belie this supposition become clear: firstly, the number of apparent open reading frames in the human genome is significantly smaller than appears to be necessary to code for all of the diverse proteins in higher organisms, and secondly that a significant proportion of the protein sequences that would be coded by the genome would not be expected to form stable three-dimensional (3D) structures. Clearly the genome must include coding for a multitude of alternative forms of proteins, some of which may be partly or fully disordered or incompletely structured in their functional states. At the same time as this likelihood was recognized, experimental studies also began to uncover examples of important protein molecules and domains that were incompletely structured or completely disordered in solution, yet remained perfectly functional. In the ensuing years, we have seen an explosion of experimental and genome-annotation studies that have mapped the extent of the intrinsic disorder phenomenon and explored the possible biological rationales for its widespread occurrence. Answers to the question 'why would a particular domain need to be unstructured?' are as varied as the systems where such domains are found. This review provides a survey of recent new directions in this field, and includes an evaluation of the role not only of intrinsically disordered proteins but also of partially structured and highly dynamic members of the disorder-order continuum.

Figure 1

Schematic figure showing (left) the domain structure of cyclic-AMP response element binding protein (CREB) binding protein (CBP) and (right) alignment of amino acid sequences of CBP from human (hs), mouse (mm), rat (rn), frog (xl) and its paralog p300 from human (hs). Amino acids are colored according to the classification acidic Glu, Asp (red); basic Lys, Arg, His (blue), hydrophobic Val, Leu, Ile, Phe, Tyr, Met (yellow), rare Cys, Trp (purple) and disorder-promoting Gly, Ala, Ser, Thr, Pro, Asn, Gln (green). Vertical black lines indicate the presence of short insertions relative to the sequence of human CBP. Aligned sequences corresponding to the structured domains denoted by spheres on the left are boxed with the corresponding color.

Figure 2

A. Schematic diagram showing domains of the tumor suppressor p53. AD: activation domain; PRD: proline-rich domain; DBD: DNA binding domain; TD: tetramerization domain; BD: C-terminal regulatory domain. B. Schematic figure illustrating the model for the interactions of p53 with CBP and HDM2 (Adapted from (Ferreon et al., 2009b)).

Figure 3

NMR ¹H-¹⁵N HSQC spectra illustrating the fast- and slow-exchange processes occurring as the KIX domain of CBP is added to the intrinsically disordered pKID domain of CREB. A. HSQC spectrum of pKID in the absence of KIX. Insets show enlarged versions of (top) the cross peak belonging to the phosphorylated Ser133 and (bottom) the cross peak of Leu138. B. superimposed HSQC spectra of pKID in the absence of KIX (blue) and in the presence of 1:0.1, 1:0.2, 1:0.3 and 1:0.4 mole ratios of KIX (colors progressing towards green). Insets show enlarged versions of the pSer133 and Leu138 cross peak sets. C. superimposed spectra for the complete titration. Corresponding assignments in the spectrum of the free and complexed pKID are linked by arrows. The pink circles represent cross peak positions calculated on the basis of the crosspeak movements illustrated in part B for the partly folded intermediate state (Sugase et al., 2007a).

Figure 4

Comparison of the structures of ligands bound to the CBP TAZ1 domain. The surface of TAZ1 (almost identical in all three complexes) is shown in gray, with the backbone of STAT2-TAD (Wojciak et al., 2009) in green, the HIF-1α-CTAD (Dames et al., 2002a) in red and the CITED2-TAD (De Guzman et al., 2004a) in blue. The left and right images represent a 180° rotation around the vertical axis in the plane of the page. The N- and C-termini of each ligand are labeled: note that STAT2 binds in the opposite sense to the other two ligands. (Adapted from (Wojciak et al., 2009))

Figure 5

Structural model of the ternary complex between the retinoblastoma protein pRb, the TAZ2 domain of CBP and the adenoviral E1A protein. The model was generated using the crystal structure of the complex of pRb with E1A (CR1, residues 37–49) (PDB entry 2R7G) (Liu and Marmorstein, 2007), the NMR structure of the complex between the TAZ2 domain of CBP and residues 53–91 of E1A (PDB entry 2KJE) (Ferreon et al., 2009c) and the crystal structure of the HPV E7 peptide (DLYCYEQLN, homologous to CR2 residues 121–129 of E1A) containing the LXCXE motif that interacts with pRb (PDB entry 1GUX) (Lee et al., 1998). The flexible linker between residues 83 and 120 of E1A is indicated schematically as a dotted line. The backbone structures of pRb, E1A and TAZ2 are represented as ribbons colored gray, coral and blue, respectively. (Adapted from (Ferreon et al., 2009c))

Figure 6

Structural comparison of the NCBD domain of CBP in complex with (left) the ACTR domain of p160 (PDB entry 1KBH) (Demarest et al., 2002) and (right) IRF3 (PDB entry 1Z0Q) (Qin et al., 2005). (Adapted from (Wright and Dyson, 2009)). Note that free ACTR is intrinsically disordered, whereas IRF3 is a globular protein in the free state.

Figure 7

A. Superposition of p53 TAD structures in various complexes, with the NCBD domain of CBP (green) (PDB entry 2L14) (Lee et al., 2010b), with MDM2 (magenta) (PDB entry 1YCQ)(Kussie et al., 1996), with RPA (red) (PDB entry 2B29) (Bochkareva et al., 2005) and with TFIIH (blue) (PDB entry 2GS0) (Di Lello et al., 2006). The corresponding residues for each complex are aligned on the backbone heavy atoms of the two well-defined helices, α1 and α2 of the NCBD complex. B. Superposition of NCBD structures in various complexes, with the p53 TAD (blue) (PDB entry 2L14) (Lee et al., 2010b), with ACTR (PDB entry 1KBH) (Demarest et al., 2002) and with SRC1 (PDB entry 2C52) (Waters et al., 2006).

Figure 8

A. Schematic diagram showing the regulation of the HIF-1α transcription factor under normal oxygenation conditions (bottom), where proline hydroxylation in the central ODD domain recruits the Von Hippel-Lindau factor, leading to degradation, and asparagine hydroxylation in the C-terminal activation domain lowers the affinity for transcriptional activators. In hypoxic conditions (top), neither the prolines not the asparagine are hydroxylated, with the result that HIF-1α is stabilized and binds to CBP/p300 to promote transcription of hypoxia-response genes. (Adapted from (Hirota and Semenza, 2005)) B. Backbone structure of one member of the family of NMR structures of the complex of HIF-1α CTAD with the TAZ1 domain of CBP (PDB entry 1L8C) (Dames et al., 2002b). The HIF-1α hydroxylation site on at Asn803 on α_B is indicated.

Figure 9

A. Single ankyrin repeat module from the Notch ankyrin repeat domain showing secondary structure elements. B. Backbone structure of the Notch ankyrin repeat domain (PDB entry 1YYH) (Ehebauer et al., 2005), showing close connections between repeat subunits.