Structure of LRRK1 and mechanisms of autoinhibition and activation.

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinson's disease while LRRK1 is linked to bone diseases. Furthermore, Parkinson's disease-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. Here, to understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

Lis1 relieves cytoplasmic dynein-1 autoinhibition by acting as a molecular wedge.

Cytoplasmic dynein-1 transports intracellular cargo towards microtubule minus ends. Dynein is autoinhibited and undergoes conformational changes to form an active complex that consists of one or two dynein dimers, the dynactin complex, and activating adapter(s). The Lissencephaly 1 gene, LIS1, is genetically linked to the dynein pathway from fungi to mammals and is mutated in people with the neurodevelopmental disease lissencephaly. Lis1 is required for active dynein complexes to form, but how it enables this is unclear. Here, we present a structure of two yeast dynein motor domains with two Lis1 dimers wedged in-between. The contact sites between dynein and Lis1 in this structure, termed 'Chi,' are required for Lis1's regulation of dynein in Saccharomyces cerevisiae in vivo and the formation of active human dynein-dynactin-activating adapter complexes in vitro. We propose that this structure represents an intermediate in dynein's activation pathway, revealing how Lis1 relieves dynein's autoinhibited state.

Structures of human dynein in complex with the lissencephaly 1 protein, LIS1.

The lissencephaly 1 protein, LIS1, is mutated in type-1 lissencephaly and is a key regulator of cytoplasmic dynein-1. At a molecular level, current models propose that LIS1 activates dynein by relieving its autoinhibited form. Previously we reported a 3.1 Å structure of yeast dynein bound to Pac1, the yeast homologue of LIS1, which revealed the details of their interactions (Gillies et al., 2022). Based on this structure, we made mutations that disrupted these interactions and showed that they were required for dynein's function in vivo in yeast. We also used our yeast dynein-Pac1 structure to design mutations in human dynein to probe the role of LIS1 in promoting the assembly of active dynein complexes. These mutations had relatively mild effects on dynein activation, suggesting that there may be differences in how dynein and Pac1/LIS1 interact between yeast and humans. Here, we report cryo-EM structures of human dynein-LIS1 complexes. Our new structures reveal the differences between the yeast and human systems, provide a blueprint to disrupt the human dynein-LIS1 interactions more accurately, and map type-1 lissencephaly disease mutations, as well as mutations in dynein linked to malformations of cortical development/intellectual disability, in the context of the dynein-LIS1 complex.

Structural basis for cytoplasmic dynein-1 regulation by Lis1.

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.

Structure Analysis of Proteins and Peptides by Difference Circular Dichroism Spectroscopy.

Difference circular dichroism (CD) spectroscopy was used here to characterize changes in structure of flexible peptides upon altering their environments. Environmental changes were introduced by binding to a large target structure, temperature shift (or concentration increase) or so-called membrane-mimicking solvents. The first case involved binding of a largely disordered peptide to its target structure associated with chromatin remodeling, leading to a transition into a highly helical structure. The second example was a short 8HD (His-Asp) repeat peptide that can bind metal ions. Both Zn and Ni at µM concentrations resulted in different type of changes in secondary structure, suggesting that these metal ions provide different environments for the peptide to assume unique secondary structures. The third case is related to a few short neuroprotective peptides that were largely disordered in aqueous solution. Increased temperature resulted in induction of significant, though small, ß-sheet structures. Last example was the induction of non-helical structures for short neuroprotective peptides by membrane-mimicking solvents, including trifluoroethanol, dodecylphosphocholine and sodium dodecylsulfate. While these agents are known to induce α-helix, none of the neuropeptides underwent transition to a typical helical structure. However, trifluoroethanol did induce α-helix for the first peptide involved in chromatin remodeling described above in the first example.

Structural insights into assembly and function of the RSC chromatin remodeling complex.

SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

Structural Insight into a Novel Formyltransferase and Evolution to a Nonribosomal Peptide Synthetase Tailoring Domain.

Nonribosomal peptide synthetases (NRPSs) increase the chemical diversity of their products by acquiring tailoring domains. Linear gramicidin synthetase starts with a tailoring formylation (F) domain, which likely originated from a sugar formyltransferase (FT) gene. Here, we present studies on an Anoxybacillus kamchatkensis sugar FT representative of the prehorizontal gene transfer FT. Gene cluster analysis reveals that this FT acts on a UDP-sugar in a novel pathway for synthesis of a 7-formamido derivative of CMP-pseudaminic acid. We recapitulate the pathway up to and including the formylation step in vitro, experimentally demonstrating the role of the FT. We also present X-ray crystal structures of the FT alone and with ligands, which unveil contrasts with other structurally characterized sugar FTs and show close structural similarity with the F domain. The structures reveal insights into the adaptations that were needed to co-opt and evolve a sugar FT into a functional and useful NRPS domain.

Piecing together nonribosomal peptide synthesis.

Nonribosomal peptide synthetases (NRPSs) produce peptide products with wide-ranging biological activities. NRPSs are macromolecular machines with modular assembly-line logic, a complex catalytic cycle, moving parts and multiple active sites. They are organized into repeating sets of domains, called modules. Each module contains all functionality to introduce a building block into the growing peptide, many also perform cosynthetic tailoring. Structures of individual domains have provided insights into their catalytic mechanisms, but with one exception, larger NRPS proteins were refractory to structure determination. Recently, structure determination succeeded for four multi-domain NRPS proteins: an alternative formylating initiation and two termination modules as well as a large cross-module construct. This review highlights how these data, together with novel didomain structures, contribute to a holistic view of the architecture, domain-domain interactions and conformational changes in NRPS megaenzymes.

Manipulation of an existing crystal form unexpectedly results in interwoven packing networks with pseudo-translational symmetry.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that synthesize a myriad of diverse molecules. Tailoring domains have been co-opted into NRPSs to introduce further variety into nonribosomal peptide products. Linear gramicidin synthetase contains a unique formylation-tailoring domain in its initiation module (F-A-PCP). The structure of the F-A di-domain has previously been determined in a crystal form which had large solvent channels and no density for the minor Asub subdomain. An attempt was made to take advantage of this packing by removing the Asub subdomain from the construct (F-AΔsub) in order to produce a crystal that could accommodate the PCP domain. In the resulting crystal the original packing network was still present, but a second network with the same packing and almost no contact with the original network took the place of the solvent channels and changed the space group of the crystal.

Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

Towards a characterization of the structural determinants of specificity in the macrocyclizing thioesterase for deoxyerythronolide B biosynthesis.

Type I polyketide synthases (PKSs) are giant multidomain proteins that synthesize many therapeutics and other natural products. The synthesis proceeds by a thiotemplate mechanism whereby intermediates are covalently attached to the PKS. The release of the final polyketide is catalyzed by the terminal thioesterase (TE) domain through hydrolysis, transesterification, or macrocyclization. The PKS 6-deoxyerythronolide B synthase (DEBS) produces the 14-membered macrolide core of the clinically important antibiotic erythromycin. The TE domain of DEBS (DEBS TE) has well-established, empirically-defined specificities for hydrolysis or macrocyclization of native and modified substrates. We present efforts towards understanding the structural basis for the specificity of the thioesterase reaction in DEBS TE using a set of novel diphenyl alkylphosphonates, which mimic substrates that are specifically cyclized or hydrolyzed by DEBS TE. We have determined structures of a new construct of DEBS TE alone at 1.7Å, and DEBS TE bound with a simple allylphosphonate at 2.1Å resolution. Other, more complex diphenyl alkylphosphonates inhibit DEBS TE, but we were unable to visualize these faithful cyclization analogs in complex with DEBS TE. This work represents a first step towards using DEBS TE complexed with sophisticated substrate analogs to decipher the specificity determinants in this important reaction.

Application of modern reversed-phase peptide retention prediction algorithms to the Houghten and DeGraw dataset: peptide helicity and its effect on prediction accuracy.

Twenty five years ago Houghten and DeGraw published a groundbreaking study of reversed-phase (RP)-HPLC retention of 298 peptide analogs, including 260 peptides coding the positional substitution in a 13-mer molecule with all 20 naturally occurring amino acids [1]. The authors challenged the state-of-the-art assumption that peptide retention can be represented as a sum of individual hydrophobicities of the constituent amino acids, and suggested an additional dependence on the ordering (sequence) of the residues. Here we explore the accuracy of modern peptide retention prediction models when applied to this retention dataset. We find that all of them perform below their claimed prediction accuracies. Clearly, the question raised 25 years ago remains unanswered, despite significant progress in the field over the past few years. Analysis of the prediction errors shows that the vast majority of outliers occur due to the amphipathic character of the framework Ac-YPYDVPDYASLRS-Amide peptide. This indicates that the understanding and quantitative description of stabilization of helical structures upon interaction with C18 phase is underdeveloped and should be a priority moving forward. In this report we also show that the presence of N-cap stabilizing residues increases peptide RP retention and should be taken into account. Capping effects have not been considered in peptide RP-HPLC studies, despite the clear evidence hidden in the quarter-century old Houghten and DeGraw's experimental results.

Effect of cyclization of N-terminal glutamine and carbamidomethyl-cysteine (residues) on the chromatographic behavior of peptides in reversed-phase chromatography.

N-terminal loss of ammonia is a typical peptide modification chemical artifact observed in bottom-up proteomics experiments. It occurs both in vivo for N-terminal glutamine and in vitro following enzymatic cleavage for both N-terminal glutamine and cysteine alkylated with iodoacetamide. In addition to a mass change of -17.03 Da, modified peptides exhibit increased chromatographic retention in reversed-phase (RP) HPLC systems. The magnitude of this increase varies significantly depending on the peptide sequence and the chromatographic condition used. We have monitored these changes for extensive sets (more than 200 each) of tryptic Gln and Cys N-terminated species. Peptides were separated on 100 Špore size C18 phases using identical acetonitrile gradient slopes with 3 different eluent compositions: 0.1% trifluoroacetic acid; 0.1% formic acid and 20 mM ammonium formate at pH 10 as ion-pairing modifiers. The observed effect of this modification on RP retention is the product of increased intrinsic hydrophobicity of the modified N-terminal residue, lowering or removing the effect of ion-pairing formation on the hydrophobicity of adjacent residues at acidic pHs; and possibly the increased formation of amphipathic helical structures when the positive charge is removed. Larger retention shifts were observed for Cys terminated peptides compared to Gln, and for smaller peptides. Also the size of the retention increase depends on the eluent conditions: pH 10≪trifluoroacetic acid