Computational Reconstruction of Multidomain Proteins Using Atomic Force Microscopy Data

SummaryClassical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures

A Generalized Allosteric Mechanism for cis-Regulated Cyclic Nucleotide Binding Domains

Cyclic nucleotides (cAMP and cGMP) regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB) domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels) using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1) the phosphate binding cassette (PBC), which binds the cAMP ribose-phosphate, 2) the “hinge,” a flexible helix, which contacts the PBC, 3) the β2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4) a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif). The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the β2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP) represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains

Refinement of Underdetermined Crystal Structures

The normal matrix for full matrix least squares contains a wealth of information about the behavior of the problem. In particular, the eigenvalues and eigenvectors contain all of the information concerning precision of parameter determination and correlation of parameters. This information is valid even if the refinement is underdetermined. Full-matrix analysis is likely to prove valuable for low resolution work as well as for high resolution work. 1 Introduction Refinement of macromolecular structures as a mathematical problem is not different from refinement of small-molecule structures. Both are straightforward optimization problems. The difficulties arise because the macromolecular crystallographer rarely has sufficient data to answer questions at the same level of detail as the small molecule crystallographer. Nevertheless, he has a lot of data, and the temptation to over-interpret it is sometimes overwhelming. As a personal note, I began work on refinement of protein structures..

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances

A binary complex of the catalytic subunit of cAMP-dependent protein kinase and adenosine further defines conformational flexibility

AbstractBackground: cAMP-dependent protein kinase (cAPK), a ubiquitous protein in eukaryotic cells, is one of the simplest members of the protein kinase family. It was the first protein kinase to be crystallized and continues to serve as a biochemical and structural prototype for this family of enzymes. To further understand the conformational changes that occur in different liganded and unliganded states of cAPK, the catalytic subunit of cAPK was crystallized in the absence of peptide inhibitor.Results: The crystal structure of the catalytic subunit of mouse recombinant cAPK (rC) complexed with adenosine was solved at 2.6 Å resolution and refined to a crystallographic R factor of 21.9% with good stereochemical parameters. This is the first structure of the rC subunit that lacks a bound inhibitor or substrate peptide. The structure was solved by molecular replacement and comprises two lobes (large and small) which contain a number of conserved loops.Conclusions: The binary complex of rC and adenosine adopts an ‘intermediate’ conformation relative to the previously described ‘closed’ and ‘open’ conformations of other rC complexes. Based on a comparison of these structures, the induced fit that is necessary for catalysis and closing of the active-site cleft appears to be confined to the small lobe, as in the absence of the peptide the conformation of the large lobe, including the peptide-docking surface, does not change. Three specific components contribute to the closing of the cleft: rotation of the small lobe; movement of the C-terminal tail; and closing of the so-called glycine-rich loop. There is no induced fit in the large lobe to accommodate the peptide and the closing of the cleft. A portion of the C-terminal tail, residues 315–334, serves as a gate for the entry or exit of the nucleotide into the hydrophobic active-site cleft