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David Baker

Publications and source records attributed to David Baker.

At least 19 recordsLinked to original sources
PubMed2025

De novo designed Hsp70 activator dissolves intracellular condensates.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70 in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of Hsp70 binding proteins that either inhibit or stimulate Hsp70 ATPase activity. An ATPase stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution and revealed roles as cell growth promoting signaling hubs. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to regulate condensates and other cellular targets.

HSP70 Heat-Shock Proteins↗
PubMed2003

An improved protein decoy set for testing energy functions for protein structure prediction.

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all-atom energy functions could identify the native and close-to-native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent-accessible surface area-based solvation provided the best enrichment and discrimination of close-to-native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X-ray crystal structures: a large energy gap was observed between native and non-native conformations for X-ray structures but not for NMR structures.

Algorithms↗
PubMed2003

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins.

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.

Amino Acid Sequence↗
PubMed2003

Deciphering a novel thioredoxin-like fold family.

Sequence--and structure-based searching strategies have proven useful in the identification of remote homologs and have facilitated both structural and functional predictions of many uncharacterized protein families. We implement these strategies to predict the structure of and to classify a previously uncharacterized cluster of orthologs (COG3019) in the thioredoxin-like fold superfamily. The results of each searching method indicate that thioltransferases are the closest structural family to COG3019. We substantiate this conclusion using the ab initio structure prediction method rosetta, which generates a thioredoxin-like fold similar to that of the glutaredoxin-like thioltransferase (NrdH) for a COG3019 target sequence. This structural model contains the thiol-redox functional motif CYS-X-X-CYS in close proximity to other absolutely conserved COG3019 residues, defining a novel thioredoxin-like active site that potentially binds metal ions. Finally, the rosetta-derived model structure assists us in assembling a global multiple-sequence alignment of COG3019 with two other thioredoxin-like fold families, the thioltransferases and the bacterial arsenate reductases (ArsC).

Amino Acid Sequence↗
PubMed2003

Conserved residue clustering and protein structure prediction.

Protein residues that are critical for structure and function are expected to be conserved throughout evolution. Here, we investigate the extent to which these conserved residues are clustered in three-dimensional protein structures. In 92% of the proteins in a data set of 79 proteins, the most conserved positions in multiple sequence alignments are significantly more clustered than randomly selected sets of positions. The comparison to random subsets is not necessarily appropriate, however, because the signal could be the result of differences in the amino acid composition of sets of conserved residues compared to random subsets (hydrophobic residues tend to be close together in the protein core), or differences in sequence separation of the residues in the different sets. In order to overcome these limits, we compare the degree of clustering of the conserved positions on the native structure and on alternative conformations generated by the de novo structure prediction method Rosetta. For 65% of the 79 proteins, the conserved residues are significantly more clustered in the native structure than in the alternative conformations, indicating that the clustering of conserved residues in protein structures goes beyond that expected purely from sequence locality and composition effects. The differences in the spatial distribution of conserved residues can be utilized in de novo protein structure prediction: We find that for 79% of the proteins, selection of the Rosetta generated conformations with the greatest clustering of the conserved residues significantly enriches the fraction of close-to-native structures.

Acyl Coenzyme A↗
PubMed2003

Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations.

Protein-protein docking algorithms provide a means to elucidate structural details for presently unknown complexes. Here, we present and evaluate a new method to predict protein-protein complexes from the coordinates of the unbound monomer components. The method employs a low-resolution, rigid-body, Monte Carlo search followed by simultaneous optimization of backbone displacement and side-chain conformations using Monte Carlo minimization. Up to 10(5) independent simulations are carried out, and the resulting "decoys" are ranked using an energy function dominated by van der Waals interactions, an implicit solvation model, and an orientation-dependent hydrogen bonding potential. Top-ranking decoys are clustered to select the final predictions. Small-perturbation studies reveal the formation of binding funnels in 42 of 54 cases using coordinates derived from the bound complexes and in 32 of 54 cases using independently determined coordinates of one or both monomers. Experimental binding affinities correlate with the calculated score function and explain the predictive success or failure of many targets. Global searches using one or both unbound components predict at least 25% of the native residue-residue contacts in 28 of the 32 cases where binding funnels exist. The results suggest that the method may soon be useful for generating models of biologically important complexes from the structures of the isolated components, but they also highlight the challenges that must be met to achieve consistent and accurate prediction of protein-protein interactions.

Algorithms↗
PubMed2003

Cannabinoids inhibit neurodegeneration in models of multiple sclerosis.

Multiple sclerosis is increasingly being recognized as a neurodegenerative disease that is triggered by inflammatory attack of the CNS. As yet there is no satisfactory treatment. Using experimental allergic encephalo myelitis (EAE), an animal model of multiple sclerosis, we demonstrate that the cannabinoid system is neuroprotective during EAE. Mice deficient in the cannabinoid receptor CB1 tolerate inflammatory and excitotoxic insults poorly and develop substantial neurodegeneration following immune attack in EAE. In addition, exogenous CB1 agonists can provide significant neuroprotection from the consequences of inflammatory CNS disease in an experimental allergic uveitis model. Therefore, in addition to symptom management, cannabis may also slow the neurodegenerative processes that ultimately lead to chronic disability in multiple sclerosis and probably other diseases.

Animals↗
PubMed2003

Protein-protein docking predictions for the CAPRI experiment.

We predicted structures for all seven targets in the CAPRI experiment using a new method in development at the time of the challenge. The technique includes a low-resolution rigid body Monte Carlo search followed by high-resolution refinement with side-chain conformational changes and rigid body minimization. Decoys (approximately 10(6) per target) were discriminated using a scoring function including van der Waals and solvation interactions, hydrogen bonding, residue-residue pair statistics, and rotamer probabilities. Decoys were ranked, clustered, manually inspected, and selected. The top ranked model for target 6 predicted the experimental structure to 1.5 A RMSD and included 48 of 65 correct residue-residue contacts. Target 7 was predicted at 5.3 A RMSD with 22 of 37 correct residue-residue contacts using a homology model from a known complex structure. Using a preliminary version of the protocol in round 1, target 1 was predicted within 8.8 A although few contacts were correct. For targets 2 and 3, the interface locations and a small fraction of the contacts were correctly identified.

Algorithms↗
PubMed2003

The effects of mutations on motions of side-chains in protein L studied by 2H NMR dynamics and scalar couplings.

Recently developed 2H spin relaxation experiments are applied to study the dynamics of methyl-containing side-chains in the B1 domain of protein L and in a pair of point mutants of the domain, F22L and A20V. X-ray and NMR studies of the three variants of protein L studied here establish that their structures are very similar, despite the fact that the F22L mutant is 3.2kcal/mol less stable. Measurements of methyl 2H spin relaxation rates, which probe dynamics on a picosecond-nanosecond time scale, and three-bond 3J(Cgamma-CO), 3J(Cgamma-N) and 3J(Calpha-Cdelta) scalar coupling constants, which are sensitive to motion spanning a wide range of time-scales, reveal changes in the magnitude of side-chain dynamics in response to mutation. Observed differences in the time-scale of motions between the variants have been related to changes in energetic barriers. Of interest, several of the residues with different motional properties across the variants are far from the site of mutation, suggesting the presence of long-range interactions within the protein that can be probed through studies of dynamics.

Bacterial Proteins↗
PubMed2003

Mifepristone or inhibition of 11beta-hydroxylase activity potentiates the sedating effects of the cannabinoid receptor-1 agonist Delta(9)-tetrahydrocannabinol in mice.

The cannabinoid system is a regulator of neurotransmission and is linked with hormonal control. We have found in experimental mouse studies that the progesterone receptor inhibitor mifepristone (RU38486, 80 mg/kg i.p.) or the 11beta-hydroxylase inhibitor metyrapone (100 mg/kg i.p.) when administered in combination with cannabinoids potentiates the transient-sedating cannabinoid receptor-1 effects of a high-dose Delta(9)-tetrahydrocannabinol (25 mg/kg i.p.), causing severe prolonged sedation associated with hypomotility, catalepsy and hypothermia. This observation has implications for human subjects taking these drugs and related compounds particularly because of the ubiquitous use of cannabis and the high potential for mifepristone and related compounds to become available on the 'black-market' as abortifacients.

Analgesics, Non-Narcotic↗
PubMed2003

Annexin II/p11 is up-regulated in Purkinje cells in EAE and MS.

The sensory neuron specific sodium channel Na(v)1.8 is normally detectable at only very low levels within cerebellar Purkinje cells. Annexin II light chain (p11) binds to the amino terminus of Na(v)1.8 and facilitates its functional expression within the cell membrane. We previously demonstrated that expression of Na(v)1.8 is up-regulated in cerebellar Purkinje cells in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS). In this study we demonstrate that expression of p11 is significantly up-regulated in Purkinje cells in EAE (71 +/- 9.0% vs 21.3 +/- 4.9% in controls) and in MS(65.5 +/- 1.6% vs 21.8 +/- 6.2% in controls). We also demonstrate a high degree of co-expression of p11 and Na(v)1.8 (84.8 +/- 8.9%). Together with earlier results which show that experimental expression of Na(v)1.8 within Purkinje cells perturbs the temporal pattern of impulse generation in these cells, our results extend the evidence for an acquired channelopathy which interferes with cerebellar function in MS.

Animals↗
PubMed2003

Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis.

Neuroinflammatory diseases, such as multiple sclerosis (MS), result from aberrant leukocyte traffic into the central nervous system (CNS). To breach the specialized blood-brain barrier, activated leukocytes interact with CNS endothelial cells (EC) and activate a CD54-mediated signaling pathway controlling the Rho GTPase. To function correctly Rho requires posttranslational prenylation, and this can be inhibited by depleting the supply of isoprenoids through inhibition of the cholesterol synthesis pathway with 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase) inhibitors (statins). Here we show that treatment of brain EC in vitro with lovastatin inhibits Rho-mediated transendothelial T cell migration. This effect can be reversed by supplementation with mevalonolactone, the downstream product of HMG-CoA reductase, or by ectopic expression of myristoylated Rho, which remains active in the absence of prenylation. In a relapsing-remitting mouse model of MS, lovastatin treatment inhibited leukocyte migration into the CNS and significantly attenuated the development of both acute and relapsing clinical disease. These studies demonstrate that the indirect pharmacological inhibition of Rho proteins in brain EC by statins can inhibit a key stage in the pathogenesis of neuroinflammation, namely leukocyte migration across the blood-brain barrier. These studies demonstrate a novel effect of statins in modulating the immune response in neuroinflammtory diseases and may provide additional rationale for their use in the treatment of MS.

Animals↗
PubMed2003

An orientation-dependent hydrogen bonding potential improves prediction of specificity and structure for proteins and protein-protein complexes.

Hydrogen bonding is a key contributor to the specificity of intramolecular and intermolecular interactions in biological systems. Here, we develop an orientation-dependent hydrogen bonding potential based on the geometric characteristics of hydrogen bonds in high-resolution protein crystal structures, and evaluate it using four tests related to the prediction and design of protein structures and protein-protein complexes. The new potential is superior to the widely used Coulomb model of hydrogen bonding in prediction of the sequences of proteins and protein-protein interfaces from their structures, and improves discrimination of correctly docked protein-protein complexes from large sets of alternative structures.

Amino Acid Sequence↗
PubMed2003

Ab initio methods.

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Computational Biology↗
PubMed2003

An autoimmune diabetes locus (Idd21) on mouse chromosome 18.

Twenty-four named Idd loci that contribute to the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse have been mapped by linkage and congenic analysis. Previously, meta-analysis of genome-wide linkage scans supported the existence of a locus for susceptibility to autoimmune phenotypes on rodent Chromosome (Chr) 18, in a position orthologous to the human typc 1 diabetes susceptibility locus IDDM6 (human Chr 18q12-q23). However, an autoimmune diabetes susceptibility locus has not previously been reported on mouse Chr 18. In this study, we demonstrate linkage of the majority of mouse Chr 18 to diabetes in a (ABH x NOD)F1 x NOD backcross. Congenic analysis, introgressing at least 92% of Biozzi ABH Chr 18 onto the NOD background, confirmed the presence of a diabetes locus. The chromosome substitution strain (NOD.ABH-Chr18) had reduced diabetes incidence compared with NOD mice (P < 0.0001). We have named the Chr 18 diabetes locus Idd21.

Alleles↗
PubMed2003

Interleukin-12 induces mild experimental allergic encephalomyelitis following local central nervous system injury in the Lewis rat.

The major pathological feature in the central nervous system (CNS) following traumatic brain injury is activation of microglia both around and distant from the injury site. Intraperitoneal administration of interleukin-12 (IL-12) after brain injury resulted in a 7% weight loss, clinical signs of mild EAE and significant myelin basic protein (MBP)-specific splenic cell proliferation. The extent of pathology, in terms of the number of inflammatory perivascular cuffs and activation of microglia was greatest if IL-12 was administered immediately compared to a week following brain injury, whether at one or two sites. Specifically immunostaining for MHC class II and iNOS on macrophages and microglia, ICAM-1 on endothelial cells and macrophages was observed around the site of injury. A degree of myelin processing was apparent from immunostaining of MBP in inflammatory cells distant from the lesion. Inflammatory cuffs comprising macrophages, activated microglia, CD4(+) T cells and iNOS(+) cells were also detected distant to the injury site in the medulla and spinal cord of animals treated with IL-12. These results suggest that immune-mediated events in which IL-12 production is stimulated as for example viral infection, superimposed on a brain injury, could provide a trigger for a MS-like pathology.

Animals↗
PubMed2003

Symmetry recognizing asymmetry: analysis of the interactions between the C-type lectin-like immunoreceptor NKG2D and MHC class I-like ligands.

Engagement of diverse protein ligands (MIC-A/B, ULBP, Rae-1, or H60) by NKG2D immunoreceptors mediates elimination of tumorigenic or virally infected cells by natural killer and T cells. Three previous NKG2D-ligand complex structures show the homodimeric receptor interacting with the monomeric ligands in similar 2:1 complexes, with an equivalent surface on each NKG2D monomer binding intimately to a total of six distinct ligand surfaces. Here, the crystal structure of free human NKG2D and in silico and in vitro alanine-scanning mutagenesis analyses of the complex interfaces indicate that NKG2D recognition degeneracy is not explained by a classical induced-fit mechanism. Rather, the divergent ligands appear to utilize different strategies to interact with structurally conserved elements of the consensus NKG2D binding site.

Amino Acids↗