Question

Intermediate filament assembly and disassembly in the cell is hypothesized to be regulated by phosphorylation. There are a number of different types of Keratin intermediate filaments in a cell. Researchers were specifically studying formation of intermediate filaments (IFs) from mixtures of “K5” and “K14” protofilaments.

a. The introduction above makes sense when you think about how intermediate filaments are formed in the cell. Explain why an intermediate filament would be composed of both K5 and K14 protofilaments using the terms primary, secondary, tertiary, and quaternary structure. Rubric (3): discussion of intermediate filament assembly includes why an IF would contain both K5 and K14 protofilaments (2). Four levels of protein structure are correctly included in discussion (1).          

Answer 1

The amino acid sequence of a specific protofilaments of IF defines the molecular structure and properties of the primary, secondary, tertiary and quaternary structures of keratins. Keratin K5/k14: It is produced together with its partner K14 as the primary keratins of the basal cells in the stratified epidermis. K14 is an essential keratin of all keratinocytes in stratified epithelia. The tail domain of K5 and K14 is required for the stabilization of keratin filaments in vitro. The heterodimers K5/K14 extensively form filaments

Primary and secondary structure

Protofilaments of IF are composed of K5 and K14, Their primary structure is alpha helices (across all species) they interact to form a coiled-coil secondary structure heterodymers and also interact individually with various other protofilaments to form keratins (This basic structure is common across all species). For example, lysine in position 23 and glutamate in position 106 are required for the proper coiled-coil secondary structure assembly of K5/K14 keratin filaments in vitro and in vivo. All proteins that form intermediate filaments have a tripartite secondary structure consisting of an N?terminal head domain, a central ??helical rod domain and C?terminal tail domain, and all proteins are able to self?assemble into filaments. The secondary structure of keratins is also divided into three parts. That is the head domain (towards the N?terminal of the molecule), the rod domain in the center and the tail domain. Each of these three domains is divided into subdomains. Domains and subdomains are determined by the amino acid sequence of the keratin and serve various functions in the assembly of keratin filaments and in the binding of keratins and keratin filaments to cell adhesion complexes or to signaling molecules. The distribution of ionic charges may vary in the different domains and subdomains, with the head and tail being positively charged.

Tertiary structure

The tertiary structure of keratins is a heterodimer that is formed by the rod domains of one acidic and one basic keratin in parallel orientation. This heterodimer is the first building block of a keratin filament. Keratin filaments are obligatory heteropolymers containing equimolar amounts of type I and type II keratins. The heteropolymeric nature of the keratins is established at the level of the double stranded coiled-coil (i.e. a heterodimer). Synthesis of keratins in the cells is tightly controlled in order to obtain stoichiometry of the acidic and basic keratin pairs and to produce the keratin filaments that are specific to particular stages of the differentiating epithelial cells. All epithelial cells at all stages of differentiation express mRNA sequences of both acidic and basic types of keratins in a coordinated fashion. Formation of a heterodimer by the parallel alignment of a single pair of type I and type II keratin is the first step in the assembly of a keratin intermediate filament. In a heterodimer, only the ??helical rod domains of the keratins align with each other and the alignment and structure are stabilized by the hydrophobic interactions of certain amino acid residues. The keratin molecules in heterodimers of soft?keratinizing?cornifying cells are aligned in parallel but slightly out of phase. This shift of about seven to eight amino acidsin the alignment allows an overlap of the head and tail of the keratins where two heterodimers come together. In contrast, the keratins in heterodimers of hard?keratinizing?cornifying cells are aligned in register and do not overlap the tail and head domains where two heterodimers come together. In vitro, heterodimers (and tetramers) are the most stable building blocks of keratin filaments. The heterodimers of keratins are still soluble in the cytoplasm but this solubility depends on the type of keratin and on the physicochemical characteristics of the cytoplasm. For example, the K8/K18 heterodimers are soluble in vitro in a medium with a pH of 9, whereas, in the same conditions, the heterodimers K5/K14 extensively form filaments.

Quaternary structure

Keratins are characterized by the capacity for forming keratin filaments with a complex quaternary structure. Formation of a tetramer: a protofilament with a diameter of 2 nm, an octamer: a protofibril (two protofilaments) with a diameter of 4.5 nm and ‘unit length filaments’ (ULFs) with a diameter of 20 nm comprising four bundled protofibrils.