Computational Design of a PAK1 Binding Protein

We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 μM. The tightest binding design, named Spider Roll, bound with an affinity of 100 μM. NMR-based structure prediction of Spider Roll based on backbone and ¹³C^β chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding ‘on-rate’ constant (? 10⁵ M^− 1 s^− 1). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes.     

Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its ∼ 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.     

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids

The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.     

Distributions of dimeric tandem repeats in non-coding and coding DNA sequences

We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast). For coding DNA, we find that all 16 distribution functions are exponential. For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function. We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms. These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations. By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer.     

Can contact potentials reliably predict stability of proteins?

The simplest approximation of interaction potential between amino acid residues in proteins is the contact potential, which defines the effective free energy of a protein conformation by a set of amino acid contacts formed in this conformation. Finding a contact potential capable of predicting free energies of protein states across a variety of protein families will aid protein folding and engineering in silico on a computationally tractable time-scale. We test the ability of contact potentials to accurately and transferably (across various protein families) predict stability changes of proteins upon mutations. We develop a new methodology to determine the contact potentials in proteins from experimental measurements of changes in protein's thermodynamic stabilities (DeltaDeltaG) upon mutations. We apply our methodology to derive sets of contact interaction parameters for a hierarchy of interaction models including solvation and multi-body contact parameters. We test how well our models reproduce experimental measurements by statistical tests. We evaluate the maximum accuracy of predictions obtained by using contact potentials and the correlation between parameters derived from different data-sets of experimental (DeltaDeltaG) values. We argue that it is impossible to reach experimental accuracy and derive fully transferable contact parameters using the contact models of potentials. However, contact parameters may yield reliable predictions of DeltaDeltaG for datasets of mutations confined to the same amino acid positions in the sequence of a single protein.     

The maturation of HIV‐1 protease precursor studied by discrete molecular dynamics

The equilibrium properties of a HIV‐1‐protease precursor are studied by means of an efficient molecular dynamics scheme, which allows for the simulation of the folding of the protein monomers and their dimerization into an active form and compare them with those of the mature protein. The results of the model provide, with atomic detail, an overall account of several experimental findings, including the NMR conformation of the mature dimer, the calorimetric properties of the system, the effects of the precursor tail on the dimerization constant, the secondary chemical shifts of the monomer, and the paramagnetic relaxation enhancement data associated with the conformations of the precursor. It is found that although the mature protein can dimerize in a unique, single way, the precursor populates several dimeric conformations in which monomers are always native‐like, but their binding can be non‐native. Proteins 2014; 82:633–639. © 2013 Wiley Periodicals, Inc.     

Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints.     

Fingerprint-based structure retrieval using electron density

We present a computational approach that can quickly search a large protein structural database to identify structures that fit a given electron density, such as determined by cryo-electron microscopy. We use geometric invariants (fingerprints) constructed using 3D Zernike moments to describe the electron density, and reduce the problem of fitting of the structure to the electron density to simple fingerprint comparison. Using this approach, we are able to screen the entire Protein Data Bank and identify structures that fit two experimental electron densities determined by cryo-electron microscopy.