Biomedical subjects

Robert A Grant

Publications and source records attributed to Robert A Grant.

8 recordsLinked to original sources

PubMed2005

Specificity versus stability in computational protein design.

Protein-protein interactions can be designed computationally by using positive strategies that maximize the stability of the desired structure and/or by negative strategies that seek to destabilize competing states. Here, we compare the efficacy of these methods in reengineering a protein homodimer into a heterodimer. The stability-design protein (positive design only) was experimentally more stable than the specificity-design heterodimer (positive and negative design). By contrast, only the specificity-design protein assembled as a homogenous heterodimer in solution, whereas the stability-design protein formed a mixture of homodimer and heterodimer species. The experimental stabilities of the engineered proteins correlated roughly with their calculated stabilities, and the crystal structure of the specificity-design heterodimer showed most of the predicted side-chain packing interactions and a main-chain conformation indistinguishable from the wild-type structure. These results indicate that the design simulations capture important features of both stability and structure and demonstrate that negative design can be critical for attaining specificity when competing states are close in structure space.

Bacterial Proteins↗

PubMed2005

Versatile modes of peptide recognition by the AAA+ adaptor protein SspB.

Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we report the crystal structure of a complex between SspB and its recognition peptide in RseA. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery.

Amino Acid Sequence↗

PubMed2005

A structural basis for 14-3-3sigma functional specificity.

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.

14-3-3 Proteins↗

PubMed2005

Consolidating critical binding determinants by noncyclic rearrangement of protein secondary structure.

We designed a single-chain variant of the Arc repressor homodimer in which the beta strands that contact operator DNA are connected by a hairpin turn and the alpha helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the sub-millisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical active-site residues in other proteins and could be a useful tool for protein design and minimization.

Bacteriophage P22↗

PubMed2004

Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA+ ClpXP protease.

The SspB adaptor enhances ClpXP degradation by binding the ssrA degradation tag of substrates and the AAA+ ClpX unfoldase. To probe the mechanism of substrate delivery, we engineered a disulfide bond between the ssrA tag and SspB and demonstrated otherwise normal interactions by solving the crystal structure. Although the covalent link prevents adaptor.substrate dissociation, ClpXP degraded GFP-ssrA that was disulfide bonded to the adaptor. Thus, crosslinked substrate must be handed directly from SspB to ClpX. The ssrA tag in the covalent adaptor complex interacted with ClpX.ATPgammaS but not ClpX.ADP, suggesting that handoff occurs in the ATP bound enzyme. By contrast, SspB alone bound ClpX in both nucleotide states. Similar handoff mechanisms will undoubtedly be used by many AAA+ adaptors and enzymes, allowing assembly of delivery complexes in either nucleotide state, engagement of the recognition tag in the ATP state, and application of an unfolding force to the attached protein following hydrolysis.

Adenosine Diphosphate↗

PubMed2004

Sculpting the proteome with AAA(+) proteases and disassembly machines.

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Structure of a designed dimeric zinc finger protein bound to DNA.

Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.

Amino Acid Sequence↗

PubMed2003

Structure of a delivery protein for an AAA+ protease in complex with a peptide degradation tag.

Substrate selection by AAA+ ATPases that function to unfold proteins or alter protein conformation is often regulated by delivery or adaptor proteins. SspB is a protein dimer that binds to the ssrA degradation tag and delivers proteins bearing this tag to ClpXP, an AAA+ protease, for degradation. Here, we describe the structure of the peptide binding domain of H. influenzae SspB in complex with an ssrA peptide at 1.6 A resolution. The ssrA peptides are bound in well-defined clefts located at the extreme ends of the SspB homodimer. SspB contacts residues within the N-terminal and central regions of the 11 residue ssrA tag but leaves the C-terminal residues exposed and positioned to dock with ClpX. This structure, taken together with biochemical analysis of SspB, suggests mechanisms by which proteins like SspB escort substrates to AAA+ ATPases and enhance the specificity and affinity of target recognition.

Adenosine Triphosphatases↗