Deciphering the Hidden Informational Content of Protein Sequences

Abstract

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe B1 in placental mammals) is variably positioned within crystal structures and exhibits 1 H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1-B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with ␤-cell dysfunction and neonatal diabetes mellitus.The efficiency of protein folding poses a fundamental problem at the intersection of biophysics, cell biology, and medicine (1, 2). Because the existence of a unique and accessible ground state is unrepresentative of polypeptides as a class of heteropolymers, foldability is an evolved property of biological sequences (3). Current kinetic models envisage a funnel-shaped free-energy landscape, enabling multiple trajectories to the native state (4 -6). What distinguishes foldable from non-foldable sequences (7), and how are bottlenecks avoided (8 -10)? The salience of these questions has been reinforced by recognition of proteotoxicity as a general pathological mechanism underlying diverse diseases (11,12). Here, we describe a cryptic folding element in a protein that is dispensable once the native state has been reached.A model is provided by insulin, a globular protein central to the regulation of vertebrate metabolism (13). Its impaired biosynthesis causes ␤-cell dysfunction and permanent neonatalonset diabetes mellitus (DM) 4 (14 -17). The insulin gene encodes a single-chain precursor, preproinsulin (Fig. 1A, top) (18). A signal peptide (gray bar) is cleaved on translocation into the endoplasmic reticulum (ER) to yield proinsulin. The precursor contains successive sequence motifs, defining B, C, and A domains (blue, black, and red , respectively, in Fig. 1A) (19). Whereas the translocated polypeptide is reduced and unfolded, oxidative folding in the ER yields a well organized A-B (insulinlike) core and disordered C-domain (dashed black segment in Fig. 1B) (20 -26). Folding is ...

Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

“…5 C ). Thus, fidelity of SP cleavage, exposing the critical proinsulin NH 2 -terminal arm, including phenylalanine-B1 ( 33 ), is crucial to normal proinsulin folding and export from the ER.…”

The Stability of Myocilin Olfactomedin Domain Variants Provides New Insight into Glaucoma as a Protein Misfolding Disorder

“…Finally, in neonatal onset diabetes mellitus, mutations that introduce an additional cysteine residue in proinsulin (30) lead to disulfide shuffling and kinetic trapping of misfolded intermediates (54). Yet, non-cysteine missense mutations in proinsulin, located in a region not even present in mature, processed, insulin, can alter folding efficiency (55). …”

INS-gene mutations: From genetics and beta cell biology to clinical disease

“…More work is still needed to understand how these point mutations affect proinsulin folding in the ER. However, it is worth noting that nearly all of these mutations are located at highly conserved residues in the preproinsulin molecule, some of which have already been shown to be important for local folding of the insulin moiety, which may help to align the B- and A-chains in order to facilitate native proinsulin disulfide pairing (Hua et al, 2006b; Liu et al, 2009, 2010c; Sohma et al, 2010; Weiss, 2009). Thus, even these mutations that do not directly alter proinsulin Cys residues are likely to perturb normal disulfide pairing of proinsulin.…”