The folding mechanism of collagen-like model peptides explored through detailed molecular simulations

Collagen, the most plentiful protein in mammals, constitutes approximately one quarter of all proteins in the human body (Stryer 1988). A variety of tissues, such as blood vessels, bone, and cartilage, owe their stability and tensile strength to collagen. The unique properties of collagen are related to its distinctive structure and amino acid composition. Unlike the compact, roughly spherical structures of globular proteins, collagen monomers are composed of three polypeptide chains that fold into a triple-helical rod (Brodsky and Persikov 2005). All side chains within this structure are solvent exposed—a stark contrast to globular structures, which have a significant number of their side chains buried within a hydrophobic core. This unique conformation imposes significant constraints on the sequence of each polypeptide chain. Given the close packing of amino acids near the common axis of the triple-helical rod, every third position in the amino acid sequence must be a Gly residue. Consequently, mature collagen sequences consist of triplets of the form X1-X2-Gly, where X1 and X2 are frequently Pro and Hyp, respectively (Stryer 1988). An additional defining characteristic of the structure of collagen is the hydrogen bonding pattern of its main chain. The backbone carbonyl of residue X1 in each triplet directly hydrogen bonds to a second polypeptide chain, and the N-H group of the Gly in each triplet hydrogen bonds to the other polypeptide chain (Brodsky and Persikov 2005). NMR and X-ray crystallographic studies on collagen-like model peptides have clarified many aspects of the mechanism of collagen folding and have been instrumental in deciphering how sequence variation affects the structure of collagen (Buevich and Baum 2001). Kinetic measurements of folding rates for collagen-like peptides suggest that folding consists of several steps, which include the formation of a triple-helical region near the C terminus—a C-terminal nucleated trimer—followed by propagation of the triple-helical structure from the C terminus to the N terminus in a “zipper-like” mechanism (Buevich et al. 2000; Buevich and Baum 2001; Bachmann et al. 2005). Bottlenecks in the folding pathway arise from two factors: (1) the formation of a C-terminal nucleated trimer, and (2) cis-trans isomerization of Gly-Pro bonds (Buevich and Baum 2001; Bachmann et al. 2005). Mutations that occur at positions normally occupied by Gly residues can alter this folding pathway and lead to the formation of structures that differ from the classic triple-helical fold. For instance, peptides containing a Gly → Ala mutation form triple-helical structures that contain a local bulge at the site of the mutation and a disruption of the normal collagen hydrogen bond pattern (Bella et al. 1994). Mutants containing amino acids with larger side chains can lead to more significant alterations in the structure (Beck et al. 2000; Buevich and Baum 2001). As a number of mutations have been linked to important connective tissue diseases, a thorough understanding of factors that affect the folding of collagen is of particular interest (Prockop and Ala-Kokko 2004). Osteogenesis imperfecta (OI) is one such connective-tissue disorder associated with missense mutations that result in substitutions for Gly residues (Rauch and Glorieux 2004). The clinical manifestations of OI vary greatly depending on the type of mutation and where it occurs in the sequence of type I collagen (Prockop and Ala-Kokko 2004). Patients with severe forms of OI suffer a myriad of symptoms, which may include multiple fractures, hearing loss, and heart failure secondary to valvular heart disease (Prockop and Ala-Kokko 2004). The most severe forms of OI are lethal in the perinatal period (Prockop and Ala-Kokko 2004). A number of studies have identified patients with both nonlethal and lethal forms of OI who have missense mutations resulting in Gly → Ser substitutions in type I collagen (Westerhausen et al. 1990; Bateman et al. 1992; Prockop and Ala-Kokko 2004). To better understand the folding mechanism of native collagen and of mutants associated with OI at an atomistic level of detail, we calculated folding trajectories of a peptide model of native collagen, (Pro-Hyp-Gly)10, which we henceforth refer to as peptide 1, and of a mutant bearing a Gly → Ser substitution that models a mutation found in α1(I) chains of some patients with OI, (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6. We refer to this latter sequence as peptide 2. Moreover, to fully explore the role that solvent has in the folding pathway, folding simulations were performed with explicit solvent. The formation of triple-helical structures from individual peptide chains occurs on a time scale that is not readily accessible from the standpoint of molecular simulations (Buevich and Baum 2001). Therefore, to simplify the calculations, we focus on the latter stages of folding using an all-trans model of peptides 1 and 2 that contains a C-terminal nucleated trimer. While this greatly reduces the computational complexity of the problem, folding to a triple-helical structure from this unfolded state likely occurs on a time scale that cannot be easily achieved using conventional molecular dynamics (MD) simulations with explicit solvent (Bachmann et al. 2005). Consequently, to promote folding within a shorter period of time, we employ a technique which enables us to observe a folding event during a MD trajectory. In the biased MD (BMD) approach, a gentle bias is introduced during MD simulations to favor the formation of a prespecified conformation, that is, the target (Harvey and Gabb 1993; Marchi and Ballone 1999; Paci and Karplus 1999). This is accomplished by (1) selecting fluctuations during a molecular simulation that take the system closer to the target conformation, and (2) by associating a small penalty to fluctuations that take the system away from the target. In the current study, we bias the trajectory, which begins with the unfolded structure, toward folded conformations. The method is attractive because the gentle bias does not significantly affect the short-term dynamics of the system and, by design, the bias is not introduced when the system undergoes fluctuations that naturally cause the protein to fold (Paci and Karplus 1999). In addition, the use of a gentle biasing function in folding simulations with explicit solvent is desirable as this helps to ensure that the solvent molecules will relax about a changing protein structure. The approach has been implemented in CHARMM and has been applied to a number of different systems (Paci and Karplus 1999; Paci et al. 2001, 2005; Morra et al. 2003). Using a combination of BMD and standard umbrella sampling, we develop a detailed picture of collagen folding that yields additional insights into the folding mechanism of collagen.