Zong Lin

GroEL-Mediated Protein Folding: Making the Impossible, Possible

ABSTRACT Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.

GroEL stimulates protein folding through forced unfolding

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroELs physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.

Visualizing Groel/Es in the Act of Encapsulating a Folding Protein

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

Triggering Protein Folding within the GroEL-GroES Complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.

Probing the conformation of the fibronectin III1-2 domain by fluorescence resonance energy transfer.

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III1 and III2, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III1–2.

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5′-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

Background: Chaperonins like the GroEL-GroES complex facilitate protein folding in the cell. Results: Substrate proteins are captured by the open, trans ring of the GroEL-ATP-GroES complex and are partially unfolded. Conclusion: Maximally efficient folding requires repeated cycles of substrate protein unfolding by the GroEL-GroES complex. Significance: Establishing how substrate proteins are processed by chaperonins is essential for understanding how proteins fold inside cells. A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.

The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-di-GMP receptor and flagella gene regulator

FleQ is an AAA+ ATPase enhancer-binding protein that regulates both flagella and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. FleQ belongs to the NtrC subfamily of response regulators, but lacks the corresponding aspartic acid for phosphorylation in the REC domain (FleQ(R), also named FleQ domain). Here, we show that the atypical REC domain of FleQ is essential for the function of FleQ. Crystal structure of FleQ(R) at 2.3Å reveals that the structure of FleQ(R) is significantly different from the REC domain of NtrC1 which regulates gene expression in a phosphorylation dependent manner. FleQ(R) forms a novel active dimer (transverse dimer), and mediates the dimerization of full-length FleQ in an unusual manner. Point mutations that affect the dimerization of FleQ lead to loss of function of the protein. Moreover, a c-di-GMP binding site deviating from the previous reported one is identified through structure analysis and point mutations.

The PilZ domain of MrkH represents a novel DNA binding motif

MrkH is the first characterized c-di-GMP related transcriptional regulator which affects type 3 fimbrial expression in response to cellular c-di-GMP level, and thus plays an important role in the biofilm formation of Klebsiella pneumoniae (Murphy and Clegg, 2012; Wilksch et al., 2011). However, how MrkH recognizes c-di-GMP and the target DNA sequence remains obscure. Here, we determine the crystal structure of MrkH/c-diGMP complex at 2.3 Å resolution. MrkH adopts a tandem two-domain structure with a canonical YcgR-N/PilZ proteins-fold, it contains two α-helixes (α1, α2) and 18 β-sheets which formed β-barrel 1 and β-barrel 2 (Fig. 1A and 1B). Our size-exclusion chromatography results demonstrate that MrkH forms a stable monomer either in the presence or absence of c-di-GMP (Fig. 1C). A DALI search for globally similar proteins revealed that MrkH/c-di-GMP has notable structural homology with PilZ domain proteins (Benach et al., 2007; Holm and Rosenstrom, 2010; Ko et al., 2010). The binding of c-di-GMP to MrkH is very similar to Pp4397 both MrkH and Pp4397 hold tightly two mutually intercalated c-di-GMP molecules, while VCA0042 combines with only one c-di-GMP (Fig. 1D). Through structure analysis we find that MrkH mainly forms H-bond with two c-di-GMP molecules (Fig. 1D and 1E). The side chains of R107 and R111 also contribute a cation–π interaction with the guanine group of C2E2 and C2E1 respectively. Multiple-sequence alignment revealed these residues are well conserved (Fig. S1). Besides the interactions between protein and ligands, two c-di-GMP molecules are also stabilized by strong base stacking interaction between mutually intercalated guanine groups. We further analyze the interactions between MrkH and c-di-GMP using ITC (Fig. S2A–G). The affinity of MrkH for c-di-GMP is high with Kd of approximately 0.24 μmol/L using the one site specific binding model(Whitney et al., 2015). MrkH also binds to c-di-GMP efficiently. However, MrkH loses the c-di-GMP binding affinity, which indicates that the connecting loop between two β-barrels is crucial for c-diGMP binding. R107 forms both H-bond and cation–π interaction with two c-di-GMPs (Fig. 1D), thus R107A mutant has greatly decreased binding affinity for c-di-GMP with a dissociation constant fifteen times higher than that of the wild-type MrkH (3.55 μmol/L vs 0.24 μmol/L). R111A mutant almost completely loses the c-di-GMP binding affinity, indicating that R111 is the most important residue for c-di-GMP binding. MrkH variants bind to c-di-GMP with an approximately stoichiometry of 1:2 (Table S1). Previous studies have demonstrated that MrkH could bind directly to the promoter of mrkHI or mrkA, and c-diGMP molecule promotes the binding of MrkH to the promoter (Tan et al., 2015; Wilksch et al., 2011; Yang et al., 2013), but it is unclear how MrkH recognizes its targets DNA. To address this problem, we incubated MrkH with various DNA fragments for EMSA. We purified recombinant MBP-MrkH, MBP-MrkH-YcgR-N domain (residues 1-104) and MBP-MrkH-PilZ domain (residues 105-end) which were incubated with the unlabeled mrkHI and mrkA promoter fragments respectively. The evident DNA-protein complex migrations were observed in lanes of MBP-MrkH and MBPMrkH-PilZ domain (Fig. 2A). It suggests that MrkH binds directly to the mrkHI and mrkA promoter sequence mainly through its PilZ domain. The EMSA also shows the migration velocity of DNA fragments constantly slow down as protein concentration increases (Fig. 2B) and MrkH-PilZ without MBP tag gives the same result (Fig. 2C). Size-exclusion chromatography analysis of PilZ domain indicates that the oligomeric state of PilZ domain is not affected by protein concentration (Fig. S3A). These observations implied that a long DNA fragment may recruit multiple PilZ domains and resulting nonspecific binding. In order to locate the DNA binding region in MrkH-PilZ domain, the vacuum electrostatics of MrkH/c-di-GMP structure is carefully analyzed. A highly positive charged hump consisting of six basic residues (R125, K127, K154, K163, K207 and R209) is identified (Fig. 1F). Since DNA is a negatively charged, this positively charged region may be critical for DNA binding. To test this hypothesis, we constructed several cognate mutants of MrkH-PilZ domain and performed EMSA with the mrkA regulatory fragment. The EMSA result shows that all these mutants lose DNA binding abilities