Trevor P. Creamer

Polyglutamine disruption of the huntingtin exon 1 N terminus triggers a complex aggregation mechanism

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTTNT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTTNT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTTNT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTTNT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both HTTNT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide and have implications for the molecular mechanism of Huntingtons disease.

Polyproline II helical structure in protein unfolded states: Lysine peptides revisited

The left‐handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven‐residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side‐chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.

Host‐guest scale of left‐handed polyproline II helix formation

Despite the clear importance of the left‐handed polyproline II (PPII) helical conformation in many physiologically important processes as well as its potential significance in protein unfolded states, little is known about the physical determinants of this conformation. We present here a scale of relative PPII helix‐forming propensities measured for all residues, except tyrosine and tryptophan, in a proline‐based host peptide system. Proline has the highest measured propensity in this system, a result of strong steric interactions that occur between adjacent prolyl rings. The other measured propensities are consistent with backbone solvation being an important component in PPII helix formation. Side chain to backbone hydrogen bonding may also play a role in stabilizing this conformation. The PPII helix‐forming propensity scale will prove useful in future studies of the conformational properties of proline‐rich sequences as well as provide insights into the prevalence of PPII helices in protein unfolded states. Proteins 2003.

Determinants of the polyproline II helix from modeling studies

Despite the fact that Tiffany and Krimm (1968a,b) formulated their hypothesis more than thirty years ago, it is only now that we are beginning to truly appreciate the importance of the PPII helical conformation. Recent experimental work has demonstrated that the polypeptide backbone possesses a significant propensity to adopt the PPII helical conformation (Kelly et al., 2001; Rucker and Creamer, 2002; Shi et al., 2002). The major determinant of this backbone propensity would appear to be backbone solvation, as was originally hinted at by Krimm and Tiffany (1974) and later suggested by a number of groups (Adzhubei and Sternberg, 1993; Sreerama and Woody, 1999; Kelly et al., 2001; Rucker and Creamer, 2002; Shi et al., 2002). The calculations and modeling described above provide data in support of this hypothesis. Each residue has its own propensity to adopt the PPII conformation, with the backbone propensity being modulated by the side chain (Kelly et al., 2001). Short, bulky side chains occlude backbone from solvent and thus disfavor the PPII conformation, while the lack of a side chain or long, flexible side chains tend to favor the conformation (Kelly et al., 2001). Again, the described calculations support this. Steric interactions (Pappu et al., 2000) and side chain conformational entropy alos contribute to the observed propensities. Furthermore, as suggested from surveys of PPII helices in proteins of known structure (Stapley and Creamer, 1999), side chain-to-backbone hydrogen bonds may well play a role in stabilizing PPII helices. The survey data, plus supporting calculations, also suggest that some polar residues may play a PPII helic-capping role analogous to that observed in alpha-helices (Presta and Rose, 1988; Aurora and Rose, 1998). Taken in sum, an atomic-level picture of the stabilization of PPII helices is beginning to emerge. Once the determinants of PPII helix formation are known in more detail, it will become possible to apply them, along with the known determinants of the alpha-helical conformation, to the understanding of protein unfolded states. If, as suggested at the beginning of this article, protein unfolded states are dominated by residues in the PPII and alpha-conformations, these data will allow for modeling of the unfolded state ensembles of specific proteins with a level of realism that has not been previously anticipated.

Left‐handed polyproline II helix formation is (very) locally driven

The left‐handed polyproline II helix (PPII) is believed to be the preferred conformation for proline‐rich regions of sequence in proteins. Such regions have been postulated to be protein‐protein interaction domains. The formation of this structure is studied here using simple Monte Carlo computer simulations employing the hard sphere potential. It is found that polyproline sequences adopt only the PPII structure in the simulations. Non‐proline, non‐glycine residues inserted as guests into polyproline host peptides are conformationally restricted by the following proline residues and tend to be part of the PPII helix. It is found through insertion of two alanine residues into polyproline that the PPII structure is not propagated through more than one non‐proline residue. This finding calls into question the hypothesis that proline‐rich regions will preferentially adopt this structure since many such sequences are comprised of less than 50% proline residues. Proteins 33:218–226, 1998.

Side‐chain conformational entropy in protein unfolded states

The largest force disfavoring the folding of a protein is the loss of conformational entropy. A large contribution to this entropy loss is due to the side‐chains, which are restricted, although not immobilized, in the folded protein. In order to accurately estimate the loss of side‐chain conformational entropy that occurs upon folding it is necessary to have accurate estimates of the amount of entropy possessed by side‐chains in the ensemble of unfolded states. A new scale of side‐chain conformational entropies is presented here. This scale was derived from Monte Carlo computer simulations of small peptide models. It is demonstrated that the entropies are independent of host peptide length. This new scale has the advantage over previous scales of being more precise with low standard errors. Better estimates are obtained for long (e.g., Arg and Lys) and rare (e.g., Trp and Met) side‐chains. Excellent agreement with previous side‐chain entropy scales is achieved, indicating that further advancements in accuracy are likely to be small at best. Strikingly, longer side‐chains are found to possess a smaller fraction of the theoretical maximum entropy available than short side‐chains. This indicates that rotations about torsions after χ2 are significantly affected by side‐chain interactions with the polypeptide backbone. This finding invalidates previous assumptions about side‐chain‐backbone interactions. Proteins 2000;40:443–450.

Structural basis for activation of calcineurin by calmodulin

The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.

Phosphorylation of Calcineurin at a novel serine-proline rich region orchestrates hyphal growth and virulence in Aspergillus fumigatus.

The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.

Side-chain entropy effects on protein secondary structure formation

Loss of conformational entropy is one of the primary factors opposing protein folding. Both the backbone and side‐chain of each residue in a protein will have their freedom of motion restricted in the final folded structure. The type of secondary structure of which a residue is part will have a significant impact on how much side‐chain entropy is lost. Side‐chain conformational entropies have previously been determined for folded proteins, simple models of unfolded proteins, α‐helices, and a dipeptide model for β‐strands, but not for polyproline II (PII) helices. In this work, we present side‐chain conformational estimates for the three regular secondary structure types: α‐helices, β‐strands, and PII helices. Entropies are estimated from Monte Carlo computer simulations. β‐Strands are modeled as two structures, parallel and antiparallel β‐strands. Our data indicate that restraining a residue to the PII helix or antiparallel β‐strand conformations results in side‐chain entropies equal to or higher than those obtained by restraining residues to the parallel β‐strand conformation. Side‐chains in the α‐helix conformation have the lowest side‐chain entropies. The observation that extended structures retain the most side‐chain entropy suggests that such structures would be entropically favored in unfolded proteins under folding conditions. Our data indicate that the PII helix conformation would be somewhat favored over β‐strand conformations, with antiparallel β‐strand favored over parallel. Notably, our data imply that, under some circumstances, residues may gain side‐chain entropy upon folding. Implications of our findings for protein folding and unfolded states are discussed. Proteins 2006.

Abundance and Distributions of Eukaryote Protein Simple Sequences

Protein simple sequences are a subclass of low complexity regions of sequence that are highly enriched in one or a few residue types. Such sequences are common in transcription regulatory proteins, in structural proteins, in proteins involved in nucleic acid interactions, and in mediating protein-protein interactions. Simple sequences of 10 or more residues, containing ≥50% of a single residue type are surveyed in this work. Both eukaryote and prokaryote proteomes are investigated with emphasis on the eukaryotes. Very large numbers of such sequences are found in all organisms surveyed. It is found that eukaryotes possess far more simple sequences per protein than do the prokaryotes. Prokaryotes display a linear relationship between number of proteins containing simple sequences and proteome size, whereas it is not clear that such a relationship holds for eukaryotes. Strikingly, it is found that each eukaryote possesses its own unique distribution of simple sequences. Within those distributions it is found that simple sequences enriched in certain residue types are clearly favored, whereas others are just as clearly discriminated against. The preferences observed are not correlated with residue occurrence. An analysis of classes of proteins of known function suggests that simple sequence occurrence and distribution may be related to protein function. Based upon this analysis, the large number of simple sequences found above that would be expected from a simple statistical model, plus the known functional importance of numerous such sequences, it is postulated that eukaryotes have evolved to not only tolerate large numbers of simple sequences but also to require them.