Crystal Structure Determination ofEscherichia coliClpP Starting from an EM-Derived Mask

Large ATP-dependent proteolytic complexes carry out the majority of intracellular proteolysis. To begin to understand the function of these proteases at a structural level, we have combined the information from a number of biophysical techniques such as electron microscopy (EM), small-angle scattering, and x-ray crystallography. In this study, we exploited the inherent symmetry ofEscherichia coliClpP, the proteolytic component of the ClpAP/XP ATP-dependent protease, to determine its x-ray crystal structure to 2.3-Å resolution starting with a phase set derived from a low-resolution mask obtained from EM and small-angle x-ray scattering analysis. Sevenfold and 14-fold noncrystallographic symmetry averaging facilitated phase extension beyond 20 Å and in combination with mask redetermination and matrix refinement was sufficient for completely determining the structure. The structure of ClpP is a homo-tetradecamer composed of two heptameric rings enclosing a cavity of ≈50 Å in diameter that compartmentalizes the 14 serine proteolytic active sites. Comparison of the ClpP structure with those of the 20S proteasome and HslV reveals a striking example of evolutionary convergence, despite them being unrelated in sequence and fold. Moreover, similarity in their overall architecture suggests a common model for their action.     

Structural switching of Staphylococcus aureus Clp protease: a key to understanding protease dynamics

ATP-dependent Clp protease (ClpP) is an attractive new target for the development of anti-infective agents. The ClpP protease consists of two heptameric rings that enclose a large chamber containing 14 proteolytic active sites. Recent studies indicate that ClpP likely undergoes conformational switching between an extended and degraded active state required for substrate proteolysis and a compacted and catalytically inactive state allowing product release. Here, we present the wild-type ClpP structures in two distinct states from Staphylococcus aureus. One structure is very similar to those solved ClpP structures in the extended states. The other is strikingly different from both the extended and the compacted state as observed in ClpP from other species; the handle domain of this structure kinks to take on a compressed conformation. Structural analysis and molecular dynamic simulations show that the handle domain predominantly controls the way in which degradation products exit the chamber through dynamic conformational switching from the extended state to the compressed state. Given the highly conserved sequences among ClpP from different species, this compressed conformation is unexpected and novel, which is potentially valuable for understanding the enzymatic dynamics and the acting mechanisms of ClpP.     

The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes

ClpP is a self-compartmentalized proteolytic assembly comprised of two, stacked, heptameric rings that, when associated with its cognate hexameric ATPase (ClpA or ClpX), form the ClpAP and ClpXP ATP-dependent protease, respectively. The symmetry mismatch is an absolute feature of this large energy-dependent protease and also of the proteasome, which shares a similar barrel-shaped architecture, but how it is accommodated within the complex has yet to be understood, despite recent structural investigations, due in part to the conformational lability of the N-termini. We present the structures of Escherichia coli ClpP to 1.9A and an inactive variant that provide some clues for how this might be achieved. In the wild type protein, the highly conserved N-terminal 20 residues can be grouped into two major structural classes. In the first, a loop formed by residues 10-15 protrudes out of the central access channel extending approximately 12-15A from the surface of the oligomer resulting in the closing of the access channel observed in one ring. Similar loops are implied to be exclusively observed in human ClpP and a variant of ClpP from Streptococcus pneumoniae. In the other ring, a second class of loop is visible in the structure of wt ClpP from E. coli that forms closer to residue 16 and faces toward the interior of the molecule creating an open conformation of the access channel. In both classes, residues 18-20 provide a conserved interaction surface. In the inactive variant, a third class of N-terminal conformation is observed, which arises from a conformational change in the position of F17. We have performed a detailed functional analysis on each of the first 20 amino acid residues of ClpP. Residues that extend beyond the plane of the molecule (10-15) have a lesser effect on ATPase interaction than those lining the pore (1-7 and 16-20). Based upon our structure-function analysis, we present a model to explain the widely disparate effects of individual residues on ClpP-ATPase complex formation and also a possible functional reason for this mismatch.     

Structural and functional insights into the chloroplast ATP-dependent Clp protease in Arabidopsis

In contrast with the model Escherichia coli Clp protease, the ATP-dependent Clp protease in higher plants has a remarkably diverse proteolytic core consisting of multiple ClpP and ClpR paralogs, presumably arranged within a dual heptameric ring structure. Using antisense lines for the nucleus-encoded ClpP subunit, ClpP6, we show that the Arabidopsis thaliana Clp protease is vital for chloroplast development and function. Repression of ClpP6 produced a proportional decrease in the Clp proteolytic core, causing a chlorotic phenotype in young leaves that lessened upon maturity. Structural analysis of the proteolytic core revealed two distinct subcomplexes that likely correspond to single heptameric rings, one containing the ClpP1 and ClpR1-4 proteins, the other containing ClpP3-6. Proteomic analysis revealed several stromal proteins more abundant in clpP6 antisense lines, suggesting that some are substrates for the Clp protease. A proteolytic assay developed for intact chloroplasts identified potential substrates for the stromal Clp protease in higher plants, most of which were more abundant in young Arabidopsis leaves, consistent with the severity of the chlorotic phenotype observed in the clpP6 antisense lines. The identified substrates all function in more general housekeeping roles such as plastid protein synthesis, folding, and quality control, rather than in metabolic activities such as photosynthesis.     

A ClpP protein model as tuberculosis target for screening marine compounds

ATP-dependent Clp protease (ClpP) is a core unit of a major bacterial protease complex employing as a new attractive drug target for that isolates, which are resistant to antibiotics. Mycobacterium tuberculosis, a gram-positive bacterium, is one of the major causes of hospital acquired infections. ClpP in Mycobacterium tuberculosis is usually tightly regulated and strictly requires a member of the family of Clp-ATPase and often further accessory proteins for proteolytic activation. Inhibition of ClpP eliminates these safeguards and start proteolytic degradation. Such uncontrolled proteolysis leads to inhibition of bacterial cell division and eventually cell death. In order to inhibit Clp protease, at first three dimensional structure model of ClpP in Mycobacterium tuberculosis was determined by comparative homology modeling program MODELLER based on crystal structure of the proteolytic component of the caseinolytic Clp protease (ClpP) from E. coli as a template protein and has 55%sequence identity with ClpP protein. The computed model's energy was minimized and validated using PROCHECK to obtain a stable model structure and is submitted in Protein Model Database (PMDB-ID: PM0075741). Stable model was further used for virtual screening against marine derived bioactive compound database through molecular docking studies using AutoDock 3.05. The docked complexes were validated and enumerated based on the AutoDock Scoring function to pick out the best marine inhibitors based on docked Energy. Thus from the entire 186 Marine compounds which were Docked, we got best 5 of them with optimal docked Energy (Ara-A: -14.31 kcal/mol, Dysinosin C: - 14.90kcal/mol, Nagelamide A: -20.49 kcal/mol, Strobilin: -8.02 kcal/mol, Manoalide: -8.81 kcal/mol). Further the five best-docked complexes were analyzed through Python Molecular Viewer software for their interaction studies. Thus from the Complex scoring and binding ability its deciphered that these Marine compounds could be promising inhibitors for ClpP as Drug target yet pharmacological studies have to confirm it.     

The Mycobacterium tuberculosis ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Mycobacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.     

Dynamics of the ClpP serine protease: A model for self-compartmentalized proteases

Abstract

ClpP is a highly conserved serine protease present in most bacterial species and in the mitochondria of mammalian cells. It forms a cylindrical tetradecameric complex arranged into two stacked heptamers. The two heptameric rings of ClpP enclose a roughly spherical proteolytic chamber of about 51 Å in diameter with 14 Ser–His–Asp proteolytic active sites. ClpP typically forms complexes with unfoldase chaperones of the AAA+ superfamily. Chaperones dock on one or both ends of the ClpP double ring cylindrical structure. Dynamics in the ClpP structure is critical for its function. Polypeptides targeted for degradation by ClpP are initially recognized by the AAA+ chaperones. Polypeptides are unfolded by the chaperones and then translocated through the ClpP axial pores, present on both ends of the ClpP cylinder, into the ClpP catalytic chamber. The axial pores of ClpP are gated by dynamic axial loops that restrict or allow substrate entry. As a processive protease, ClpP degrades substrates to generate peptides of about 7–8 residues. Based on structural, biochemical and theoretical studies, the exit of these polypeptides from the proteolytic chamber is proposed to be mediated by the dynamics of the ClpP oligomer. The ClpP cylinder has been found to exist in at least three conformations, extended, compact and compressed, that seem to represent different states of ClpP during its proteolytic functional cycle. In this review, we discuss the link between ClpP dynamics and its activity. We propose that such dynamics also exist in other cylindrical proteases such as HslV and the proteasome.     

Molecular Mechanism of Polypeptide Translocation Catalyzed by the Escherichia coli ClpA Protein Translocase

The removal of damaged or unneeded proteins by ATP-dependent proteases is crucial for cell survival in all organisms. Integral components of ATP-dependent proteases are motor proteins that unfold stably folded proteins that have been targeted for removal. These protein unfoldases/polypeptide translocases use ATP to unfold the target proteins and translocate them into a proteolytic component. Despite the central role of these motor proteins in cell homeostasis, a number of important questions regarding the molecular mechanisms of enzyme catalyzed protein unfolding and translocation remain unanswered. Here, we demonstrate that Escherichia coli ClpA, in the absence of the proteolytic component ClpP, processively and directionally steps along the polypeptide backbone with a kinetic step size of ∼ 14 amino acids, independent of the concentration of ATP with a rate of ∼ 19 amino acids s⁻¹ at saturating concentrations of ATP. In contrast to earlier studies by others, we have developed single-turnover fluorescence stopped-flow methods that allow us to quantitatively examine the molecular mechanism of the motor component ClpA decoupled from the proteolytic component ClpP. For the first time, we reveal that in the absence of ClpP ClpA translocates polypeptides directionally, processively and in discrete steps similar to other motor proteins that translocate vectorially on a linear lattice, such as nucleic acid helicases and kinesin. We believe that the methods employed here will be generally applicable to the examination of other AAA?+ protein translocases involved in a variety of important biological functions where the substrate is not covalently modified; for example, membrane fusion, membrane transport, protein disaggregation, and protein refolding.     

Crystallography and mutagenesis point to an essential role for the N-terminus of human mitochondrial ClpP

We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.     

Distinctive types of ATP-dependent Clp proteases in cyanobacteria

Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis and are thought to be ancestors to plant chloroplasts. Like chloroplasts, cyanobacteria possess a diverse array of proteolytic enzymes, with one of the most prominent being the ATP-dependent Ser-type Clp protease. The model Clp protease in Escherichia coli consists of a single ClpP proteolytic core flanked on one or both ends by a HSP100 chaperone partner. In comparison, cyanobacteria have multiple ClpP paralogs plus a ClpP variant (ClpR), which lacks the catalytic triad typical of Ser-type proteases. In this study, we reveal that two distinct soluble Clp proteases exist in the unicellular cyanobacterium Synechococcus elongatus. Each protease consists of a unique proteolytic core comprised of two separate Clp subunits, one with ClpP1 and ClpP2, the other with ClpP3 and ClpR. Each core also associates with a particular HSP100 chaperone partner, ClpC in the case of the ClpP3/R core, and ClpX for the ClpP1/P2 core. The two adaptor proteins, ClpS1 and ClpS2 also interact with the ClpC chaperone protein, likely increasing the range of protein substrates targeted by the Clp protease in cyanobacteria. We also reveal the possible existence of a third Clp protease in Synechococcus, one which associates with the internal membrane network. Altogether, we show that presence of several distinctive Clp proteases in cyanobacteria, a feature which contrasts from that in most other organisms.     

Solution structure of heavy meromyosin by small-angle scattering

Elucidation of x-ray crystal structures for the S1 subfragment of myosin afforded atomic resolution of the nucleotide and actin binding sites of the enzyme. The structures have led to more detailed hypotheses regarding the mechanisms by which force generation is coupled to ATP hydrolysis. However, the three-dimensional structure of double-headed myosin consisting of two S1 subfragments has not yet been solved. Therefore, to investigate the overall shape and relative orientations of the two heads of myosin, we performed small-angle x-ray and neutron scattering measurements of heavy meromyosin containing all three light chains (LC(1-3)) in solution. The resulting small-angle scattering intensity profiles were best fit by models of the heavy meromyosin head-tail junction in which the angular separation between heads was less than 180 degrees. The S1 heads of the best fit models are not related by an axis of symmetry, and one of the two S1 heads is bent back along the rod. These results provide new information on the structure of the head-tail junction of myosin and indicate that combining scattering measurements with high resolution structural modeling is a feasible approach for investigating myosin head-head interactions in solution.     

The ATPase ClpX is conditionally involved in the morphological differentiation of <Emphasis Type="Italic"> Streptomyces lividans</Emphasis>

ATP-dependent proteases of the ClpP type are widespread in eubacteria. These proteolytic complexes are composed of a proteolytic subunit and an ATPase subunit. They are involved in the degradation of denatured proteins, but also play a role in specific regulatory pathways. In Streptomyces lividans strains which lack the proteolytic subunit ClpP1, cell cycle progression has been shown to be blocked at early stages of growth. In this study, we examined the role of the ATPase subunit ClpX, a possible partner of the products of the clpP1 operon. A clpX mutant was obtained and it was shown that its growth was impaired only on acidic medium. Thus, the clpX phenotype differs from the clpP1 phenotype, indicating that these two components have only partially overlapping roles. We also analyzed the expression of clpX. Although clpX expression is increased under heat-shock conditions in many bacteria, we found that this is not the case in S. lividans.     

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.     

Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site

ClpP, the proteolytic component of the ATP-dependent ClpAP and ClpXP chaperone/protease complexes, has 14 identical subunits organized in two stacked heptameric rings. The active sites are in an interior aqueous chamber accessible through axial channels. We have determined a 1.9 A crystal structure of Escherichia coli ClpP with benzyloxycarbonyl-leucyltyrosine chloromethyl ketone (Z-LY-CMK) bound at each active site. The complex mimics a tetrahedral intermediate during peptide cleavage, with the inhibitor covalently linked to the active site residues, Ser97 and His122. Binding is further stabilized by six hydrogen bonds between backbone atoms of the peptide and ClpP as well as by hydrophobic binding of the phenolic ring of tyrosine in the S1 pocket. The peptide portion of Z-LY-CMK displaces three water molecules in the native enzyme resulting in little change in the conformation of the peptide binding groove. The heptameric rings of ClpP-CMK are slightly more compact than in native ClpP, but overall structural changes were minimal (rmsd approximately 0.5 A). The side chain of Ser97 is rotated approximately 90 degrees in forming the covalent adduct with Z-LY-CMK, indicating that rearrangement of the active site residues to a active configuration occurs upon substrate binding. The N-terminal peptide of ClpP-CMK is stabilized in a beta-hairpin conformation with the proximal N-terminal residues lining the axial channel and the loop extending beyond the apical surface of the heptameric ring. The lack of major substrate-induced conformational changes suggests that changes in ClpP structure needed to facilitate substrate entry or product release must be limited to rigid body motions affecting subunit packing or contacts between ClpP rings.     

ATP-dependent association between subunits of Clp protease in pea chloroplasts

The chloroplast ATP-dependent Clp protease (EC 3.4.21.92) is composed of the proteolytic subunit ClpP and the regulatory ATPase, ClpC. Although both subunits are found in the stroma, the interaction between the two is dynamic. When immunoprecipitation with antibodies against ClpC was performed on stroma from dark-adapted pea (Pisum sativum L. cv. Alaska) chloroplasts, ClpC but not ClpP was precipitated. However, when stroma was supplemented with ATP, both ClpC and ClpP were precipitated. Co-immunoprecipitation was even more efficient in the presence of ATP-gamma-S, suggesting that the association between regulatory and proteolytic subunits is dependent on binding of ATP to ClpC, but not its hydrolysis. To further test this association, stroma was fractionated by column chromatography, and the presence of Clp subunits in the different fractions was monitored immunologically. When stroma depleted of ATP was fractionated on an ion-exchange column, ClpP and ClpC migrated separately, whereas in the presence of ATP-gamma-S both subunits co-migrated. Similar results were observed in size-exclusion chromatography. To further characterize the precipitated enzyme, its proteolytic activity was assayed by testing its ability to degrade beta-casein. No degradation was observed in the absence of ATP, and degradation was inhibited in the presence of phenylmethylsulfonyl fluoride, consistent with Clp being an ATP-dependent serine protease. The activity of the isolated enzyme was further tested using chimeric OE33 as a model substrate. This protein was also degraded in an ATP-dependent manner, supporting the suggested role of Clp protease as a major housekeeping protease in the stroma.     

Scanning X-Ray Nanodiffraction on Dictyostelium discoideum

We have performed scanning x-ray nanobeam diffraction experiments on single cells of the amoeba Dictyostelium discoideum. Cells have been investigated in 1), freeze-dried, 2), frozen-hydrated (vitrified), and 3), initially alive states. The spatially resolved small-angle x-ray scattering signal shows characteristic streaklike patterns in reciprocal space, which we attribute to fiber bundles of the actomyosin network. From the intensity distributions, an anisotropy parameter can be derived that indicates pronounced local variations within the cell. In addition to nanobeam small-angle x-ray scattering, we have evaluated the x-ray differential phase contrast in view of the projected electron density. Different experimental aspects of the x-ray experiment, sample preparation, and data analysis are discussed. Finally, the x-ray results are correlated with optical microscopy (differential phase contrast and confocal microscopy of mutant strains with fluorescently labeled actin and myosin II), which have been carried out in live and fixed states, including optical microscopy under cryogenic conditions.     

Human mitochondrial ClpP is a stable heptamer that assembles into a tetradecamer in the presence of ClpX

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.     

Mycobacterium tuberculosis ClpP Proteases Are Co-transcribed but Exhibit Different Substrate Specificities

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.     

Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the ¹⁵N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.